CN103429606A - Engineered nucleic acids and methods of use thereof - Google Patents

Engineered nucleic acids and methods of use thereof Download PDF

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CN103429606A
CN103429606A CN2011800575554A CN201180057555A CN103429606A CN 103429606 A CN103429606 A CN 103429606A CN 2011800575554 A CN2011800575554 A CN 2011800575554A CN 201180057555 A CN201180057555 A CN 201180057555A CN 103429606 A CN103429606 A CN 103429606A
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nucleic acid
protein
cell
cells
nucleotide sequence
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贾森·施伦
格雷戈里·J·西兹克维克兹
凯内齐·伊杰贝
萨伊达·尔巴施尔
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现代治疗公司
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Priority to US61/404,413 priority
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Priority to PCT/US2011/054636 priority patent/WO2012045082A2/en
Publication of CN103429606A publication Critical patent/CN103429606A/en

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Abstract

Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the use of modified messenger RNAs, and are useful for production of proteins.

Description

设计核酸及其使用方法 Design of nucleic acids and methods of use

[0001] 相关申请的交叉引用 CROSS [0001] REFERENCE TO RELATED APPLICATIONS

[0002] 本申请要求2010年10月I日提交的美国临时申请61/404,413的优先权,其公开的内容视为本申请公开内容的一部分(并在此通过引用而全文引入)。 [0002] This application claims the United States in October 2010 I filed provisional application No. 61 / 404,413, the disclosure of which is considered part of the present disclosure is (and is hereby incorporated by reference).

背景技术 Background technique

[0003] 天然存在的RNA是由四种基本的核糖核苷酸ATP、CTP、UTP和GTP合成的,但也可以含有转录后修饰的核苷酸。 [0003] Naturally occurring RNA nucleotide long chains of four ribonucleotides ATP, CTP, UTP and GTP synthesis, but may also contain post-translational modifications of nucleotides. 而且,在RNA中已经鉴定出大约100种不同的核苷修饰(Rozenski,J,Crain,P,和McCloskey, J(1999)。RNA 修饰数据库:1999 年更新,Nucl AcidsRes 27:196-197)。 Moreover, the RNA has identified about 100 different nucleoside modifications (Rozenski, J, Crain, P, and McCloskey, J (1999) .RNA modified database: 1999 update, Nucl AcidsRes 27: 196-197). 但是,在免疫刺激潜能和RNA翻译效率方面,核苷修饰所起的作用尚不明了。 However, the immune stimulation potential and RNA translation efficiency, a modified nucleoside role is unclear.

[0004] 影响蛋白质表达的现有方法学中存在许多问题。 [0004] There are many problems in the conventional method of learning affect protein expression. 例如,引入细胞的异源DNA (不论异源DNA是否整合入染色体)可能由子代或后代细胞遗传。 For example, a heterologous DNA introduced into a cell (whether heterologous DNA integrated into the chromosome) may be inherited by the progeny cells or progeny. 引入的DNA可以以某种频率整合入宿主细胞的基因组DNA,从而改变和/或破坏宿主细胞的基因组DNA。 Introduced DNA may be integrated into the genome of the host cell DNA at a certain frequency, to change and / or destruction of the genomic DNA of the host cell. 此外,在产生蛋白质之前必须经过多个步骤。 Further, the protein must be produced before a plurality of steps. 一旦进入细胞,DNA必须转运到细胞核中,在细胞核内转录成RNA。 Once inside the cells, DNA must be transported into the nucleus, the nucleus is transcribed into RNA. 之后从DNA转录的RNA必须进入细胞质,在细胞质内翻译成蛋白质。 After RNA DNA from the cytoplasm to be transcribed, translated into proteins in the cytoplasm. 对多个处理步骤的需求导致了在感兴趣的蛋白质产生之前的时间延滞。 The need for multiple processing steps results in a time lag before the protein of interest produced. 此外,在细胞内实现DNA的表达存在困难;经常地,DNA进入细胞但没有表达,或者没有以合理的速度或浓度表达。 Further, there are difficulties to achieve intracellular expression of the DNA; often, but no expression of DNA into the cell, or not expressed at a reasonable rate or concentration. 将DNA引入诸如原代细胞或修饰的细胞系的细胞时,这可能尤其是个问题。 When introducing DNA into cells such as primary cells or cell lines modified, it may be a particular problem.

[0005] 本领域需要解决核调节酸细胞内翻译的生物学模式。 [0005] The need in the art to solve the nuclear regulatory biological model for translation in cells acids.

[0006] 除非另有解释,否则在本文中使用的所有技术和科学术语与本发明所属技术领域普通技术人员通常的理解具有相同的含义。 [0006] Unless otherwise explained, all technical and scientific terms used herein and in the art of the present invention is commonly understood by one of ordinary skill have the same meaning. 尽管,类似于或等同于本文所述的方法和材料也可用于本发明的实施和检测,但在此描述了适合的方法与材料。 Despite, similar or equivalent methods and materials described herein can be used in the practice and testing of the present invention, but are described herein, suitable methods and materials. 材料、方法和实施例仅为示例性的,而非旨在限制。 The materials, methods and examples are illustrative only, and not intended to be limiting. 本发明其他特征可以自以下发明详述和权利要求明显看出。 Other features of the present invention may be from the following detailed description and the appended claims the invention apparent.

[0007] 发明简述 [0007] SUMMARY OF THE INVENTION

[0008] 本文描述了生产蛋白质、多肽和肽的方法。 [0008] Described herein are a method of producing proteins, polypeptides and peptides. 例如,所述方法包括在所述感兴趣的蛋白质、多肽或肽在细胞内产生(例如,翻译)的条件下,将编码感兴趣的蛋白质、多肽或肽的核酸(例如,本文所述的修饰核酸)引入细胞(例如,人细胞)。 For example, the method includes generating a modified (e.g., translation) under conditions of a protein nucleic acid, encoding a polypeptide or peptide of interest (e.g., as described herein in a cell in a protein, polypeptide or peptide of interest nucleic acid) introduced into a cell (e.g., human cells). 在一些具体实施方式中,所述核酸包含一个或多个核苷修饰(例如,一个或多个本文所述的核苷修饰)。 In some embodiments, the nucleic acid comprises one or more modified nucleosides (e.g., one or more nucleosides modified as described herein). 在一些具体实施方式中,所述核酸能逃脱该核酸所引入的细胞的天然免疫反应。 In some embodiments, the nucleic acid can escape the natural immune response of the nucleic acid introduced cells. 在一些具体实施方式中,所述蛋白质、多肽或肽是本文所述的治疗性蛋白质。 In some embodiments, the protein, polypeptide or peptide is a therapeutic protein described herein. 在一些具体实施方式中,所述蛋白质、多肽或肽包含一个或多个翻译后修饰(例如,人细胞中存在的翻译后修饰)。 In some embodiments, the protein, polypeptide or peptide comprising one or modification (e.g., post-translational modifications present in human cells) after a plurality of translation. 本文还描述了用以生产蛋白质的组合物和试剂盒。 Also described herein are compositions and kits for the production of proteins. 本文进一步描述了具有(例如,通过本文所述的方法产生的)改变的蛋白质水平的细胞和培养物。 This paper further describes a protein level (e.g., produced by the methods described herein) and altered cell cultures.

[0009] 一方面,本发明涉及在细胞内生产感兴趣的蛋白质(例如,异源蛋白质)的方法,所述方法包括步骤:(i )提供能进行蛋白质翻译的靶细胞;以及(ϋ )在所述感兴趣的蛋白质在靶细胞内生产的条件下,向靶细胞内引入包含第一分离的核酸的组合物,所述第一分离的核酸包含编码感兴趣的蛋白质的可翻译区和核苷修饰。 [0009] In one aspect, the present invention relates to the production of a protein of interest in a cell (e.g., a heterologous protein), the method comprising the steps of: (i) providing a target cell capable of protein translation; and (ϋ) in under the conditions of protein production in target cells of interest, introducing a first composition comprising an isolated nucleic acid into a target cell, comprising a first isolated nucleic acid encoding a protein of interest may untranslated region and nucleotides modification. 在一些实施方式中,所述方法进一步包括从细胞中充分纯化感兴趣的蛋白质的步骤。 In some embodiments, the method further comprises the step of substantially purified protein of interest from the cell. 在一些实施方式中,所述感兴趣的蛋白质是分泌蛋白。 In some embodiments, the protein of interest is a secreted protein.

[0010] 另一方面,本发明涉及在细胞内生产感兴趣的蛋白质(例如,异源蛋白质)的方法,所述方法包括步骤:(i )提供能进行蛋白质翻译的靶细胞,以及(ii )在所述感兴趣的蛋白质在细胞内产生的条件下,向靶细胞内引入组合物,所述组合物包含:(a)包含编码感兴趣的蛋白质的可翻译区和核苷修饰的第一分离的核酸;和(b)包含抑制性核酸的第二核酸。 [0010] In another aspect, the present invention relates to the production of a protein of interest in a cell (e.g., a heterologous protein), the method comprising the steps of: (i) providing a protein translation capable of target cells, and (ii) under the conditions of the protein of interest produced within a cell, a target cell is introduced into the composition, said composition comprising: (a) a first separation of interest comprising a protein coding region can be translated and modified nucleosides nucleic acids; and (b) a second nucleic acid comprises an inhibitory nucleic acid. 在一些实施方式中,所述方法进一步包括从细胞中充分纯化感兴趣的蛋白质的步骤。 In some embodiments, the method further comprises the step of substantially purified protein of interest from the cell. 在一些具体实施方式中,所述感兴趣的蛋白质是分泌蛋白。 In some embodiments, the protein of interest is a secreted protein.

[0011] 一方面,本发明涉及提高细胞内感兴趣的重组表达蛋白质的产量的方法,包括步骤:(i )提供包含编码感兴趣的蛋白质的重组核酸的靶细胞;和(ii )在效应蛋白(effector protein)在祀细胞内生产的条件下,向祀细胞引入含有第一分离的核酸的组合物,从而提高细胞内重组表达蛋白的产量,所述第一分离的核酸包含编码翻译效应蛋白的可翻译区和核苷修饰。 [0011] In one aspect, the present invention relates to a method of increasing interest within cells expressing the recombinant production of protein, comprising the steps of: (i) providing a target cell comprising a recombinant nucleic acid of interest encoding a protein; and (ii) in the effector proteins (effector protein) under the conditions of the Si produced in the cell, introducing a composition comprising a first isolated nucleic acid to the Si cell, thereby increasing the intracellular production of recombinant protein expression, the isolated nucleic acid encoding a first effector proteins translated translatable region and nucleoside modifications.

[0012] 在一些具体实施方式中,所述靶细胞是哺乳动物细胞。 [0012] In some embodiments, the target cells are mammalian cells. 在一些具体实施方式中,所述靶细胞是酵母细胞。 In some embodiments, the target cell is a yeast cell. 在一些具体实施方式中,所述靶细胞是细菌细胞、昆虫细胞或植物细胞。 In some embodiments, the target cells are bacterial cells, insect cells or plant cells. 在一些具体实施方式中,所述感兴趣的蛋白质是分泌蛋白质。 In some embodiments, the protein of interest is a secreted protein. 在一些具体实施方式中,所述感兴趣的蛋白质是跨膜蛋白。 In some embodiments, the protein of interest is a transmembrane protein. 在一些具体实施方式中,所述感兴趣的蛋白质是抗体或其抗原结合片段。 In some embodiments, the protein of interest is an antibody or antigen-binding fragment thereof. 在一些具体实施方式中,所述感兴趣的蛋白质是生长因子或细胞因子。 In some embodiments, the protein of interest is a growth factor or a cytokine. 在一些具体实施方式中,所述感兴趣的蛋白质是肽或肽类似物。 In some embodiments, the protein of interest is a peptide or peptide analog. 在一些具体实施方式中,所述翻译效应蛋白是神经酰胺转移蛋白(CERT)。 In some embodiments, the protein is a translation effector ceramide transfer protein (CERT). 在一些具体实施方式中,在靶细胞中以有效提高重组表达蛋白质翻译效率的量翻译所述翻译效应蛋白。 In some embodiments, the target cells in an amount effective to increase expression of recombinant protein translation efficiency of translation of the effector protein translation. 在一些具体实施方式中,在靶细胞中以有效减少细胞内除了重组表达蛋白质以外的蛋白质的翻译效率的量翻译所述翻译效应蛋白。 In some embodiments, the target cells effective to reduce translation of the translation effector protein translation efficiency of intracellular protein amount other than the expression of recombinant proteins in addition. 在一些具体实施方式中,在靶细胞中以有效减少含有重组表达蛋白质的包涵体的形成的量翻译所述翻译效应蛋白。 In some embodiments, the target cell an amount effective to reduce formation of inclusion bodies containing the recombinant expression of the protein translated protein translation effector. 在一些具体实施方式中,在靶细胞中以有效减少细胞内重组表达蛋白质的降解的量翻译所述翻译效应蛋白。 In some embodiments, the target cells effective to reduce expression of the intracellular degradation of the recombinant protein translated in the translation effector proteins. 在一些具体实施方式中,在靶细胞中以有效增加重组表达蛋白质的分泌的量翻译所述翻译效应蛋白。 In some embodiments, the target cells effective to increase expression of recombinant protein translation effect secretion of the translated protein.

[0013] 另一方面,本发明涉及用以改变靶细胞中感兴趣的蛋白质的水平的方法,所述方法包括步骤:(i )调节靶细胞内的至少一种翻译效应分子的活性;以及(ϋ )培养所述细胞。 [0013] In another aspect, the present invention relates to a method for varying the level of protein in the target cell of interest, said method comprising the steps of: (i) modulating the activity of at least one translation effector molecule in a target cell; and ( ϋ) culturing the cells. 在一些具体实施方式中,所述靶细胞不包含重组核酸。 In some embodiments, the target cell does not comprise recombinant nucleic acid. 在一些具体实施方式中,所述方法进一步包括分离感兴趣的蛋白质的步骤。 In some embodiments, the method further comprising the step of separating the protein of interest.

[0014] 另一方面,本发明涉及用以调整靶细胞内感兴趣的蛋白质水平的方法,包括步骤:i )调节靶细胞内至少一种翻译效应分子的活性,其中所述调节包括向靶细胞中引入含有 [0014] In another aspect, the present invention relates to a method for adjusting the level of the protein of interest within a target cell, comprising steps: i) modulating the activity of at least one translation effector molecules within a target cell, wherein said adjusting comprises a target cell introducing containing

编码翻译效应蛋白的可翻译区和核苷修饰的第一分离的核酸;以及ii )培养所述细胞。 Encoding the effector protein translation may be translated region and a first modified nucleoside isolated nucleic acid; and ii) culturing the cell.

[0015] 一方面,本发明涉及一种具有改变了的蛋白质水平的动物细胞(例如,哺乳动物细胞),其通过下述步骤产生:(i )向细胞内引入有效量的含有编码翻译效应蛋白的可翻译区和核苷修饰的第一分离的核酸;以及(ϋ )培养所述细胞。 [0015] In one aspect, the present invention relates to an animal cell (e.g., mammalian cells) having an altered level of protein, which is produced by the following steps: (i) introducing an effective amount of a protein effector which encodes the translation into the cell the translated region can be modified nucleoside and a first isolated nucleic acid; and (ϋ) culturing the cells. 在某些实施方式中,引入细胞的第一分离的核酸的有效量通过滴定由可翻译区翻译的蛋白质的预期量确定。 In certain embodiments, the effective amount of nucleic acid introduced into a cell a first isolated was determined by titration translated by a desired amount of protein translated regions.

[0016] 一方面,本发明涉及含有大量本发明所述细胞的高密度培养物。 [0016] In one aspect, the present invention relates to a high-density culture containing a large number of the cells of the invention. 在一些具体实施方式中,所述培养包括分批补料过程。 In some embodiments, the culture comprises a fed-batch process. 在一些具体实施方式中,所述培养包括连续给料过程。 In some embodiments, the culture comprises a continuous feeding process.

[0017] 一方面,本发明涉及用于蛋白质生产的组合物,所述组合物包含含有可翻译区和核苷修饰的第一分离的核酸,以及适合第一核酸的可翻译区翻译的哺乳动物细胞,其中所述核酸显示出减少的由细胞核酸酶引起的降解。 [0017] In one aspect, the present invention relates to compositions for protein production, the mammal comprises a nucleic acid composition comprising a first isolated region can be translated and modified nucleosides, and suitable nucleic acid may be first translated regions Translation cells, wherein said nucleic acid exhibits degradation by cellular nucleases reduced. 在一些具体实施方式中,所述哺乳动物细胞含有重组核酸。 In some embodiments, the mammalian cell comprises a recombinant nucleic acid.

[0018] 另一方面,本发明涉及用于蛋白质生产的组合物,所述组合物包含:(i )含有可翻译区和核苷修饰的第一分离的核酸,其中所述核酸显示出减少的由细胞核酸酶引起的降解;(ϋ )含有抑制性核酸的第二核酸;以及(iii)适合第一核酸的可翻译区翻译的哺乳动物细胞,其中所述哺乳动物细胞含有能与抑制性核酸反应的目标核酸。 [0018] another aspect, the present invention relates to compositions for the production of proteins, said composition comprising: (i) containing a modified nucleoside untranslated region and a first isolated nucleic acid, wherein said nucleic acid exhibits reduced caused by the degradation of cellular nucleases; (ϋ) inhibitory nucleic acid containing a second nucleic acid; and (iii) for the first nucleic acid may be translated regions translated mammalian cell, wherein said mammalian cell comprising a nucleic acid capable of inhibiting the reaction of the target nucleic acid. 在一些具体实施方式中,所述哺乳动物细胞含有重组核酸。 In some embodiments, the mammalian cell comprises a recombinant nucleic acid.

[0019] 一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括包含可翻译区和核酸修饰的第一分离的核酸,及其包装和说明书,其中所述核酸能逃脱所述第一分离的核酸所引入的细胞的天然免疫反应。 [0019] In one aspect, the present invention relates to a kit for producing a protein, the kit comprising a nucleic acid comprising a modified translation region and a first isolated nucleic acid, its packaging and instructions, wherein said nucleic acid can escape the innate immune response of the first nucleic acid is introduced into isolated cells.

[0020] 另一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括:(i )以引入靶细胞时有效产生预期量的由可翻译区编码的蛋白质的量提供的,含有可翻译区的第一分离的核酸;(ϋ )以有效地基本抑制细胞天然免疫反应的量提供的含有抑制性核酸的第二核酸;以及(iii)其包装和说明书。 [0020] another aspect, the present invention relates to a kit for producing a protein, the kit comprising: (i) when introduced into the target cells in an effective amount to produce the desired amount of protein provided by encoding the translated region, comprising the isolated nucleic acid may be first translated region; amount (ϋ) effective to substantially inhibit cellular innate immune response provided by the second nucleic acid comprises an inhibitory nucleic acid; and (iii) its packaging and instructions.

[0021] 另一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括含有可翻译区和核苷修饰的第一分离的核酸,及其包装和说明书,其中所述核酸显示出减少的由细胞核酸酶引起的降解。 [0021] In another aspect, the present invention relates to a kit for protein production, the kit comprises a translated region containing a first nucleoside modification and isolated nucleic acid, its packaging and instructions, wherein said nucleic acid exhibits reduction caused by degradation of cellular nucleases.

[0022] 一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括含有可翻译区和至少二个不同核苷修饰的第一分离的核酸,及其包装和说明书,其中所述核酸显示出减少的由细胞核酸酶引起的降解。 [0022] In one aspect, the present invention relates to a kit for protein production, the kit comprises a translated region and containing at least two different nucleoside modifications of a first isolated nucleic acid, its packaging and instructions, wherein said It shows the nucleic acid degradation by cellular nucleases reduced.

[0023] 另一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括:(i )含有可翻译区的第一分离的核酸;(ϋ )含有抑制性核酸的第二核酸;以及(iii)其包装和说明书。 [0023] In another aspect, the present invention relates to a kit for producing a protein, the kit comprising: (i) a first isolated nucleic acid comprising a translatable region; (ϋ) a second nucleic acid comprising an inhibitory nucleic acid; and (iii) its packaging and instructions.

[0024] 另一方面,本发明涉及用于蛋白质生产的试剂盒,所述试剂盒包括:(i )含有可翻译区和至少一个核苷修饰的第一分离的核酸,其中所述核酸显示出减少的由细胞核酸酶引起的降解;(ϋ )含有抑制性核酸的第二核酸;以及(iii)其包装和说明书。 [0024] In another aspect, the present invention relates to a kit for producing a protein, the kit comprising: (i) untranslated region and containing at least one nucleoside modified first isolated nucleic acid, wherein said nucleic acid exhibits reduction caused by the degradation of cellular nucleases; (ϋ) a second nucleic acid comprising an inhibitory nucleic acid; and (iii) its packaging and instructions.

[0025] 一方面,本发明涉及用于蛋白质生产的试剂盒,包括编码可翻译区的第一分离的核酸,以及其包装和说明书,其中可翻译区编码蛋白质,其中所述第一核酸含有核酸修饰,其中所述第一核酸与不含核酸修饰的核酸相比,在所述第一分离的核酸所引入的细胞中的降解显示出减少。 [0025] In one aspect, the present invention relates to a kit for producing a protein, comprising a first isolated nucleic acid encoding a translation region, as well as packaging and instructions, which can be translated region encoding a protein, wherein said first nucleic acid comprising a nucleic acid modification, wherein the first nucleic acid as compared to no nucleic acid modified nucleic acid degradation in a cell of said first isolated nucleic acid is introduced exhibit reduced.

[0026] 另一方面,本发明涉及用于蛋白质生产的试剂盒,包括编码可翻译区的第一分离的核酸,以及其包装和说明书,其中可翻译区编码蛋白质,其中所述第一核酸含有核酸修饰,其中所述第一核酸与不含核酸修饰的核酸相比,在所述第一分离的核酸所引入的细胞中显不出更稳定。 [0026] another aspect, the present invention relates to a kit for producing a protein, comprising a first isolated nucleic acid encoding a translation region, as well as packaging and instructions, which can be translated region encoding a protein, wherein said first nucleic acid comprising nucleic acid modification, wherein the first nucleic acid as compared to no nucleic acid modified nucleic acid in cells of said first isolated nucleic acid introduced in nearly as good as more stable.

[0027] —方面,本发明涉及用于免疫球蛋白生产的试剂盒,包括第一分离的核酸,以及包装和说明书,其中所述第一分离的核酸包含:i )编码免疫球蛋白的可翻译区和ii )核酸修饰,其中所述第一核酸能逃脱所述第一分离的核酸所引入的细胞的天然免疫反应,其中所述可翻译区基本不含胞苷和尿嘧啶核苷酸。 [0027] - aspect, the present invention relates to a kit for producing immunoglobulins, comprising a first isolated nucleic acid, as well as packaging and instructions, wherein the first isolated nucleic acid comprising: i) encoding an immunoglobulin translatable region and ii) nucleic acid modification, wherein the first nucleic acid can escape the innate immune response of the isolated nucleic acid introduced into the first cell, wherein said region is substantially free of a cytidine translation and uracil nucleotides.

[0028] 另一方面,本发明涉及通过使用上述试剂盒产生的哺乳动物细胞。 [0028] another aspect, the present invention relates to mammalian cells by using the above kit.

[0029] 另一方面,本发明涉及自含有第一分离的核酸的生产细胞产生的分离的免疫球蛋白,所述第一分离的核酸含有i )编码免疫球蛋白的可翻译区和ϋ)核酸修饰,其中所述第一分离的核酸能逃脱所述细胞的天然免疫反应,其中所述可翻译区基本不含胞苷或尿嘧啶核苷酸或胞苷和尿嘧啶核苷酸的组合。 [0029] another aspect, the present invention relates to an isolated immunoglobulin comprising a first isolated nucleic acid from the production cells, the first isolated nucleic acid comprising i) encodes an immunoglobulin and can be translated region ϋ) a nucleic acid modification, wherein the first isolated nucleic acid can escape the innate immune response of the cell, wherein said composition is substantially free of the translation region or uracil nucleotides cytidine or cytidine and uridine nucleotides.

[0030] 一方面,本发明涉及含有有效量的本文所述蛋白质的药物制剂。 [0030] In one aspect, the present invention relates to a pharmaceutical formulation comprising an effective amount of the protein described herein.

[0031] 另一方面,本发明涉及含有有效量的第一核酸的药物制剂,所述第一核酸含有i )编码免疫球蛋白的可翻译区和ii )核酸修饰,其中所述第一核酸显示出减少的由细胞核酸酶引起的降解且能逃脱所述第一核酸所引入的细胞的天然免疫反应,其中所述可翻译区基本不含胞苷和尿嘧啶核苷酸。 [0031] another aspect, the present invention relates to a pharmaceutical formulation comprising an effective amount of a first nucleic acid, said first nucleic acid comprising i) encoding the immunoglobulin may be translated regions and ii) a nucleic acid modification, wherein the first nucleic acid displays the reduction caused by the degradation of cellular nucleases and can escape from the innate immune response of the first nucleic acid introduced into a cell, wherein said region is substantially free of a cytidine translation and uracil nucleotides.

[0032] 前述方法、细胞、培养物、组合物、制剂和试剂盒的具体实施方式可以包含一个或多个以下特征: [0032] The foregoing method, cell cultures, compositions, formulations and kits of the specific embodiments may comprise one or more of the following features:

[0033] 在一些具体实施方式中,所述第一分离的核酸包括信使RNA (mRNA)。 [0033] In some embodiments, the first isolated nucleic acid comprising messenger RNA (mRNA). 在一些具体实施方式中,所述mRNA包括选自以下组成组的至少一个核苷:B密唳-4-酮核苷、5-氮-尿苷、2-硫-5-氮-尿苷、2-硫代尿苷、4-硫-假尿苷、2-硫-假尿苷、5-羟基尿苷、3-甲基尿苷、5-羧甲基-尿苷、1-羧甲基-假尿苷、5-炔丙基-尿苷、1-炔丙基-假尿苷、5-牛磺酸甲基尿苷、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫-尿苷、1-牛磺酸甲基-4-硫-尿苷、5-甲基-尿苷、1-甲基-假尿苷、4-硫-1-甲基-假尿苷、2-硫-1-甲基-假尿苷、1-甲基_1_去氮_假尿苷、2-硫-1-甲基-1-去氮_假尿苷、二氢尿苷、二氢假尿苷、2_硫-二氢尿苷、2-硫-二氢假尿苷、2-甲氧基尿苷、2-甲氧基-4-硫-尿苷、4-甲氧基-假尿苷和4-甲氧基-2-硫-假尿苷。 In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: B-tight Li 4-one nucleosides, 5-N - uridine, 2-thio -5- N ​​- uridine, 2- thiouridine, 4-thiouracil - pseudouridine, 2-thio - pseudouridine, 5-hydroxy-uridine, 3-methyl-uridine, 5-carboxymethyl - uridine, 1-carboxymethyl - pseudouridine, 5-propargyl - uridine, 1-propargyl - pseudouridine, 5-methyluridine taurine, methyl taurine 1- - pseudouridine, 5-taurocholate sulfuric acid-methyl-2 - uridine, 1-methyl-4-taurine sulfur - uridine, 5-methyl - uridine, 1-methyl - pseudouridine, 4-thiouracil-1- yl - pseudouridine, 1-methyl-2-thio - pseudouridine, 1-methyl-deaza _1_ _ pseudouridine, 2-methyl-1-deaza sulfur _ pseudouridine , dihydrouridine, pseudouridine dihydro, sulfur 2_ - dihydro-uridine, 2-thio - dihydro pseudouridine, 2-methoxy-uridine, 2-methoxy-4 sulfur - Urine glycosides, 4-methoxy - pseudouridine sulfur and 4-methoxy-2 - pseudouridine. 在一些具体实施方式中,所述mRNA包括选自以下组成组的至少一个核苷:5_氮-胞苷、伪异胞苷、3-甲基-胞苷、N4-乙酰基胞苷、5-甲酰基基胞苷、N4-甲基胞苷、5-羟甲基胞苷、1-甲基-伪异胞苷、吡咯并胞苷、吡咯并伪异胞苷、2-硫_胞苷、2-硫-5-甲基-胞苷、4-硫_伪异胞苷、4-硫-1-甲基-伪异胞苷、4-硫-1-甲基-1-去氮-伪异胞苷、1-甲基-1-去氮-伪异胞苷、zebularine、5_氮-zebularine、5_甲基-zebularine、5_ 氮-2-硫-zebularine、2_ 硫-zebularine、2_ 甲氧基-胞苷、2-甲氧基-5-甲基-胞苷、4-甲氧基-伪异胞苷和4-甲氧基-1-甲基-伪异胞苷。 In some embodiments, the at least one mRNA nucleosides selected from the group comprising: nitrogen 5_ - cytidine, pseudo-isocytidine, 3-methyl - cytidine, cytidine N4-acetyl, 5 - formyl group cytidine, N4-methyl cytidine, 5-hydroxymethyl cytidine, 1-methyl - pseudo iso cytidine, cytidine pyrrolo, pyrrolo pseudo-isocytidine, 2-thio cytidine _ , 2-thio-5-methyl - cytidine, pseudo-isocytidine _ 4-thiouracil, 4-thiouracil-1-methyl - pseudo-isocytidine, 4-methyl-1-deaza sulfur - pseudo-isocytidine, 1-methyl-1-deaza - pseudo-isocytidine, zebularine, 5_ nitrogen -zebularine, 5_ methyl -zebularine, 5_ nitrogen -2- sulfur -zebularine, 2_ sulfur -zebularine, 2_ methoxy - cytidine, 2-methoxy-5-methyl - cytidine, 4-methoxy - pseudo-isocytidine and 4-methoxy-1-methyl - pseudo-isocytidine. 在一些具体实施方式中,所述mRNA包括选自以下组成组的至少一个核苷:2_氨基嘌呤、2,6-二氨基嘌呤、7-去氮-腺嘌呤、7-去氮-8-氮-腺嘌呤、7-去氮-2-氨基嘌呤、7-去氮-8-氮-2-氨基嘌呤、7-去氮-2,6-二氨基嘌呤、7-去氮-8-氮-2,6-二氨基嘌呤、1-甲基腺苷、N6-甲基腺苷、N6-异戊烯基腺苷、N6-(顺式羟基异戊烯基)腺苷、2-甲硫基-N6-(顺式羟基异戊烯基)腺苷、N6-甘氨酰氨基甲酰腺苷、N6-苏氨酰氨基甲酰腺苷、2-甲硫基-N6-苏氨酰氨基甲酰腺苷、N6,N6-二甲基腺苷、7-甲基腺嘌呤、2-甲硫基-腺嘌呤、和2-甲氧基-腺嘌呤。 In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 2_ aminopurine, 2,6-diaminopurine, 7-deaza - adenine, 7-deaza -8- N - adenine, 7-deaza-2-amino purine, 7- deaza-aza-2-amino purine, 7-deaza-2,6-diaminopurine, 7-deaza-aza 2,6-diaminopurine, 1-methyl adenosine, N6-methyl adenosine, N6-isopentenyl adenosine, N6-(cis-hydroxy-isopentenyl) adenosine, 2-methylthio group -N6- (cis-hydroxy-isopentenyl) adenosine, N6-glycylamino carboxamido adenosine, N6-threonyl acylamino carboxamido adenosine, 2-methylthio-threonyl acylamino -N6- carboxamido adenosine, N6, N6- adenosine dimethyl, 7-methyl-adenine, 2-methylthio-- adenine, and 2-methoxy - adenine. 在一些具体实施方式中,mRNA包括选自以下组成组的至少一个核苷:肌苷、1-甲基-肌苷、丫苷、怀丁昔(wybutosine)、7_去氣_鸟昔、7_去氣-8-氣-鸟昔、6_硫-鸟昔、6_硫-7-去氣_鸟苷、6-硫-7-去氮-8-氮-鸟苷、7-甲基-鸟苷、6-硫-7-甲基-鸟苷、7-甲基肌苷、6-甲氧基鸟苷、1-甲基鸟苷、N2-甲基鸟苷、N2,N2-二甲基鸟苷、8-氧-鸟苷、7-甲基-8-氧-鸟昔、1_甲基_6-硫_鸟昔、N2-甲基_6-硫_鸟昔和N2, N2- 甲基_6-硫_鸟昔。 In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: inosine, 1-methyl - inosine, Ah glycosides, Whiting celecoxib (wybutosine), 7_ degassing _ past the birds, 7 _ -8- gas to gas - birds past, 6_ sulfur - birds past, 6_ sulfur degassing _ -7- guanosine, 6-aza deaza sulfur -7- - guanosine, 7-methyl - guanosine, 7-methyl-6- sulfur - guanosine, 7-methyl inosine, guanosine 6-methoxy-1-methyl guanosine, N2- methyl guanosine, N2, N2- two methyl guanosine, 8-oxo - guanosine, 7-methyl-8-oxo - Xi birds, birds _ 1_ methyl _6- Xi sulfur, sulfur N2- methyl _6- _ birds celecoxib and N2, N2- methyl _6- sulfur _ birds past.

附图说明 BRIEF DESCRIPTION

[0034] 图1描述了用编码人G-CSF的modRNA体外转染中国仓鼠卵巢后12和24小时测量的人G-CSF酶联免疫实验(ELISA)的条形图。 [0034] Figure 1 describes a modRNA encoding human G-CSF in vitro transfection after 12 and 24 hours measurements Chinese hamster ovary bar human G-CSF enzyme linked immunosorbent assay (ELISA) a.

[0035] 图2描述了用编码曲妥珠单抗(Trastuzumab)的modRNA的重链和轻链体外转染中国仓鼠卵巢细胞后12、24和36小时的人IgG的酶联免疫实验(ELISA)的条形图。 [0035] FIG 2 depicts transfection 12, 24 and 36 hours with Chinese hamster ovary cells in vitro heavy and light chain encoding modRNA trastuzumab (of Trastuzumab) human IgG enzyme-linked immunosorbent assay (ELISA) the bar graph.

[0036] 图3描述了用编码曲妥珠单抗的modRNA的重链和轻链体外转染人胚肾细胞(HEK293)后36小时测量的人IgG的酶联免疫实验(ELISA)的条形图。 [0036] Figure 3 depicts a bar measured after 36 hours using heavy and light chain encoding vitro trastuzumab modRNA of transfected human embryonic kidney cells (an HEK293) human IgG enzyme-linked immunosorbent assay (ELISA) of Fig. RU R2、R3是在24孔板中进行的三次转染实验且通过未处理样品校正。 RU R2, R3 is carried out in three 24-well plates and transfected with experimental calibration by the untreated sample.

[0037] 图4描述了用编码曲妥珠单抗的modRNA的重链和轻链体外转染中国仓鼠卵巢细胞后24小时测定的western blot图像。 [0037] FIG 4 depicts a western blot assay 24 hours after the image with the heavy and light chain encoding vitro trastuzumab modRNA transfection of Chinese hamster ovary cells. HC和LC分别表示曲妥珠单抗的重链和轻链。 HC and LC denote trastuzumab heavy and light chains.

[0038] 图5描述了用编码曲妥珠单抗和利妥昔单抗(Rituximab)两者的modRNA的重链和轻链体外转染中国仓鼠卵巢细胞后13小时的细胞免疫染色的图像。 [0038] FIG 5 depicts an image transfected Chinese hamster ovary cells after 13 hours with the heavy and light chains in vitro trastuzumab modRNA encoding music and Rituximab (of Rituximab) both immunostained cells.

[0039] 图6描述了编码曲妥珠单抗和利妥昔单抗的modRNA的结合免疫印迹实验的图像。 [0039] FIG 6 depicts western blot image encoding binding trastuzumab and rituximab in the modRNA. 黑框表示感兴趣的蛋白。 Black box represents the protein of interest.

[0040] 发明详述 [0040] DETAILED DESCRIPTION

[0041] 本文描述了生产蛋白质、多肽和肽的方法。 [0041] Described herein are a method of producing proteins, polypeptides and peptides. 本发明至少部分地提供了在细胞内生产感兴趣的蛋白质、多肽或肽(例如,异源蛋白质)的方法,提高细胞内感兴趣的蛋白质、多肽或肽(例如,重组表达蛋白质)的产量的`方法,以及改变细胞内感兴趣的蛋白质、多肽或肽的水平的方法。 The present invention provides at least part of the protein, polypeptide or peptide of interest produced in a cell (e.g., heterologous protein) of the method, increasing the protein, polypeptide or peptide of interest within cells (e.g., recombinant proteins) Yield `method, and a method of changing the level of a protein, polypeptide or peptide within a cell of interest. 例如,所述方法可以包括在所述感兴趣的蛋白质、多肽或肽在细胞内生产(例如,翻译)的条件下,将编码感兴趣的蛋白质、多肽或肽的核酸(例如,本文所述的修饰核酸)引入细胞(例如,人细胞)的步骤。 For example, the method may include producing (e.g., translation) under the conditions of interest encoding a protein, polypeptide or peptide nucleic acids (e.g., as described herein in a cell in a protein, polypeptide or peptide of interest modified nucleic acid) into cells (e.g., human cells) step. 在一些具体实施方式中,所述核酸含有一个或多个核苷修饰(例如,本文所述的一个或多个核苷修饰)。 In some embodiments, the nucleic acid comprises one or more modified nucleosides (e.g., one or more nucleosides modified as described herein). 在一些具体实施方式中,所述核酸包括一个或多个核苷修饰(例如,一个或多个本文所述的核苷修饰)。 In some embodiments, the nucleic acid comprises one or more modified nucleosides (e.g., a modified nucleoside of the one or more herein). 在一些具体实施方式中,所述核酸能逃脱所述核酸所引入的细胞的天然免疫反应,从而提高细胞内蛋白生产效率。 In some embodiments, the nucleic acid can escape the innate immune response cells of the nucleic acid introduced, thereby improving the efficiency of the intracellular protein production. 在一些具体实施方式中,所述蛋白质是本文所述的治疗性蛋白质。 In some embodiments, the protein is a therapeutic protein described herein. 在一些具体实施方式中,所述蛋白质含有一个或多个翻译后修饰(例如,存在于人类细胞中的翻译后修饰)。 In some embodiments, the protein comprises one or more post-translational modifications (e.g., human cells are present in the translational modification). 本文还描述了用于蛋白质生产的组合物和试剂盒。 Also described herein are compositions and kits for protein production. 本文进一步描述了具有(例如,通过本文所述方法产生的)改变的蛋白质水平的细胞和培养物。 This paper further describes a protein level (e.g., produced by the methods described herein) and altered cell cultures.

[0042] 通常,引入细胞内的外源核酸(特别是病毒核酸)引起天然免疫反应,从而导致产生干扰素(IFN)和细胞死亡。 [0042] Generally, introduction of an exogenous nucleic acid (especially viral nucleic acid) in the innate immune response due to the cell, resulting in the production of interferon (IFN) and cell death. 尽管如此,对于重组蛋白质的生产,人们仍感兴趣于将核酸(例如核糖核酸(RNA))递送入细胞(例如,在细胞培养物中、在体外、在体内或离体)从而在细胞内翻译该核酸和生产编码的蛋白质。 Nevertheless, for the production of recombinant proteins, there is still interest in a nucleic acid (e.g. RNA (an RNA)) delivered into cells (e.g., in cell culture, in vitro, in vivo or ex vivo) such that translation within the cell and production of proteins encoded by the nucleic acid. 本文部分地提供了编码能调节细胞功能和/或活性的有用的多肽的核酸,以及制备和使用这些核酸和多肽的方法。 This article provides a coding function of cells and / or nucleic acids useful activity of the polypeptide, and methods of making and using these nucleic acids and polypeptides can be adjusted. 如本文所述,这些核酸能减少其所引入的细胞群的天然免疫活性,因而提高蛋白质在该细胞群中的生产效率。 As described herein, these nucleic acids can reduce the activity of innate immune cell populations which they are introduced, thereby improving the efficiency of protein production in the cell population. 进一步地,本文描述了本发明所述核酸和蛋白质的一个或多个额外的有利活性和/或性质。 Further, it described herein a nucleic acid and protein of the present invention is advantageous or more additional active and / or properties.

[0043] 蛋白质生产方法 [0043] The method of protein production

[0044] 本文提供的方法可用于提高细胞培养过程中蛋白质产物的产量。 [0044] The methods provided herein can be used to increase production in cell culture protein products. 相对于相应的未修饰核酸,在含有大量宿主细胞的细胞培养物中,本文所述的修饰的mRNA的引入导致蛋白质生产效率提高。 Relative to the corresponding non-modified nucleic acid in a host cell culture containing a large amount of cells, the introduction of the modified mRNA as described herein results in protein production efficiency. 例如通过表现出细胞转染的增加、自所述核酸翻译的蛋白质的增加、核酸降解的减少和/或所述宿主细胞天然免疫反应的减少,可以证明蛋白质生产效率的这种提高。 For example, by increasing the transfected cells exhibited increased from the nucleic acid of the translated protein, nucleic acid degradation is reduced and reduction / or innate immune response of the host cell, can prove that a protein increase productivity. 可以通过ELISA测定蛋白质的生产,可以通过本领域已知的多种功能试验测定蛋白质活性。 Production of proteins can be determined by ELISA, various functions by known test measures the activity of a protein of the present art. 可以在连续补料或分批补料的过程中产生所述蛋白质产物。 The protein product may be produced during the continuous fed-batch or fed in.

[0045] 细胞培养和生长 [0045] Cell culture and growth

[0046] 在本发明的方法中,培养细胞。 [0046] In the method of the present invention, the cells are cultured. 细胞可以悬浮培养或贴壁培养。 Cells may be suspension culture or adherent culture. 细胞可以在多种容器(包括,例如生物反应器、细胞袋、摇袋(wave bag)、培养皿、烧瓶、超级烧瓶(hyperflasks)和其他本领域普通技术人员已知的容器)中培养。 Cells can be in a variety of containers (including, for example, a bioreactor, cell bags, shake the bag (wave bag), petri dishes, flasks, super flask (hyperflasks) and others known to those of ordinary skill in the vessel) the culture. 细胞可以在IMDMdnvitrogen,目录编号12440-53)或包括化学上确定的培养基配方在内的任意其他合适的培养基中培养。 Any other suitable media for cell number 12440-53 may be in IMDMdnvitrogen, directory), or comprising a chemically defined medium formulations including cultured. 对本领域普通技术人员而言,适合细胞培养的周围环境条件(例如温度和大气组成)是熟知的。 To those of ordinary skill in the art, for cell culture ambient conditions (e.g., temperature and atmospheric composition) are well known. 本发明的方法可用于任何适于在蛋白质生产中使用的细胞。 The method of the present invention may be used in any cell suitable for use in protein production. 一个具体实施方式中,所述细胞选自由动物细胞(例如,哺乳动物细胞)、细菌细胞、植物、微生物、藻类和真菌细胞组成的组。 A particular embodiment, the cells are selected from the group consisting of an animal cell (e.g., mammalian cells), bacterial cells, group of plants, microbes, algae and fungal cells. 在一些具体实施方式中,所述细胞是哺乳动物细胞,例如人类、小鼠、大鼠、山羊、马、兔、仓鼠或牛细胞。 In some embodiments, the cells are mammalian cells, such as human, mouse, rat, goat, horse, rabbit, hamster or bovine cells. 例如,所述细胞可以来自任何已建立的细胞系,包括但不限于HeLa、NSO、SP2/0、HEK 293T、Vero、Caco、Caco-2、MDCK、C0S-1、C0S-7、K562、Jurkat, CHO-KU DG44、CHOKISV, CHO-S, Huvec, CV-U HuH_7、NIH3T3、HEK293、293、A549, HepG2, MR-90、MCF-7、U-20S、Per.C6、SF9、SF21 或中国仓鼠卵巢细胞(CHO)。 For example, the cells may be from any established cell lines, including but not limited to, HeLa, NSO, SP2 / 0, HEK 293T, Vero, Caco, Caco-2, MDCK, C0S-1, C0S-7, K562, Jurkat , CHO-KU DG44, CHOKISV, CHO-S, Huvec, CV-U HuH_7, NIH3T3, HEK293,293, A549, HepG2, MR-90, MCF-7, U-20S, Per.C6, SF9, SF21, or China hamster ovary cells (CHO). 某些具体实施方式中,所述细胞是真菌细胞,例如选自由黄孢菌(Chrysosporium)细胞、曲霉(Aspergillus)细胞、木霉(Trichoderma)细胞、网柱黏菌(Dictyostelium)细胞、假丝酵母(Candida)细胞、酵母(Saccharomyces)细胞、粟酒裂殖酵母(Schizosaccharomyces)细胞和青霉(Penicillium)细胞组成的组的细胞。 In certain specific embodiments, the cell is a fungal cell, for example, selected from the group consisting of yellow cinerea (Chrysosporium) cells, Aspergillus (Aspergillus) cells, Trichoderma (Trichoderma) cell, the net post slime mold (Dictyostelium) cells, Candida (Candida) cells, yeast (Saccharomyces) cells, group of cells pombe (Schizosaccharomyces) cells and Penicillium (Penicillium) cells. 在某些其他具体实施方式中,所述细胞是细菌细胞,例如大肠杆菌、枯草芽孢杆菌或BL21细胞。 In certain other embodiments, the cell is a bacterial cell, such as E. coli, Bacillus subtilis or BL21 cells. 用于本方法转染的初级和次级细胞可以自多种组织获得,且包括所有能在培养中维持的细胞种类。 Primary and secondary cells may be from a variety of tissue used in the method of transfection is obtained, and includes all types of cells can be maintained in culture. 例如,通过本方法转染的初级和次级细胞可以包括成纤维细胞、角化细胞、上皮细胞(例如,乳腺上皮细胞、肠上皮细胞)、内皮细胞、神经胶质细胞、神经细胞、血液组成成分(例如,淋巴细胞、骨髓细胞)、肌肉细胞和上述体细胞种类的前体细胞。 For example, by the process of primary and secondary transfection cells may include fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, blood composition component (e.g., lymphocytes, bone marrow cells), muscle cells and somatic type of the precursor cells. 初级细胞可以自同一物种或另一物种供体(例如,小鼠、大鼠、兔、猫、狗、猪、牛、鸟、绵羊、山羊、马)获得。 Primary cells can be obtained from the same donor species or another species (eg, mice, rats, rabbits, cats, dogs, pigs, cows, birds, sheep, goats, horses).

[0047] 本发明的细胞可用于能纯化和通过常规给药方式递送的治疗性产品的体外生产。 [0047] The cells of the invention can be used for purification and in vitro production of a conventional mode of administration by delivery of therapeutic products. 无论是否扩大,这些细胞均能用于收获细胞内或细胞外蛋白质产物的大规模培养。 Whether expansion, these cells can be used within a large scale culture cells are harvested cells or extracellular protein products.

[0048] 细胞核酸递送的方法 [0048] The nucleic acid delivery methods Cell

[0049] 本发明的方法增强了核酸在体内、离体或在培养物中向细胞群中的递送。 [0049] The method of the present invention enhances the nucleic acid in vivo, ex vivo or in culture were delivered to the cell population. 例如,将包含大量宿主细胞(例如真核细胞,例如酵母或哺乳动物细胞)的细胞培养物与含有增强的核酸的组合物相接触,所述增强的核酸具有至少一个核苷修饰和任选的可翻译区。 For example, a host cell containing a large amount (e.g. eukaryotic cells, such as yeast or mammalian cells) is contacted with a cell culture composition comprising a nucleic acid enhanced, the enhancement of the nucleic acid having at least one modified nucleoside and optionally translatable region. 所述组合物还通常含有转染试剂或其他提高增强的核酸向宿主细胞中的摄取效率的化合物。 The compositions also generally contain a compound to a host cell uptake efficiency transfection reagent or other reinforcement to improve the nucleic acid. 相对于相应的未修饰核酸,所述增强的核酸在细胞群中显示出增强的保留能力。 Relative to a corresponding non-modified nucleic acids, said nucleic acid exhibits enhanced retention capacity increased in the cell population. 所述增强的核酸的保留能力比未修饰核酸的保留能力高。 The enhanced high retention capacity nucleic than unmodified nucleic acid retention capacity. 在一些具体实施方式中,比未修饰的核酸的保留能力至少高50%、75%、90%、95%、100%、150%、200%或超过200%。 In some embodiments, the ratio of the unmodified nucleic acid retention capacity is at least 50%, 75%, 90%, 95%, 100%, 150%, 200%, or more than 200%. 这种保留能力的优势可以通过用增强的核酸进行一轮转染获得或者通过后续多轮重复转染获得。 The advantage of this ability to retain or obtain can be obtained by subsequent repeated rounds of transfection by a round of transfection with nucleic acids enhanced. [0050] 向细胞中引入修饰的或瞬时的RNA用于蛋白质生产 [0050] introduced into cells or modified RNA was used for transient protein production

[0051] 瞬时转染的细胞可通过本领域普通技术人员熟知的转染、电穿孔、阳离子介质、聚合物或基于脂质的递送分子的方法产生。 [0051] The transiently transfected cells are well known by those of ordinary skill in the art transfection, electroporation, cationic medium molecular polymer or lipid-based delivery method of generating. 如果合适,可以在传统分批式步骤或连续流加(continuous flow through)步骤中,将所述修饰的瞬时RNA引入培养的细胞中。 If appropriate, a step may be a conventional batch or continuous flow plus (continuous flow through) step, the modified instantaneous RNA into cultured cells. 本发明的方法和组合物可用于制备具有提高的一个或多个感兴趣蛋白质产量的细胞。 The methods and compositions of the present invention may be used in the preparation of cells of interest having improved production of a protein of one or more. 可以用一个或多个RNA转染细胞或者将其引入细胞。 RNA may be with one or more transfected cells or introduced into cells thereof. 可以用二个或更多个RNA构建体同时或相继转染细胞。 It may be two or more simultaneously or sequentially RNA construct transfected cells. 在某些具体实施方式中,可以多次使用本文所述方法以获得具有提高的一个或多个感兴趣的RNA或蛋白质的表达的细胞。 In certain embodiments, the methods described herein may be used a plurality of times to obtain a cell expressing an RNA or protein of interest having one or more of enhanced. 例如,可以使用一个或多个编码感兴趣的RNA或蛋白质的RNA构建体转染细胞,并按照本文所述方法进行分离。 For example, RNA of interest may use one or more proteins or RNA encoded by the construct transfected cells, and isolated according to the methods described herein. 所述分离的细胞可以随后进一步地用于一个或多个编码感兴趣的RNA或蛋白质的其他RNA转染并再次分离。 The other RNA isolated cells may then be further interest for one or more proteins or RNA encoded by transfection and separated again. 例如,所述方法可用于产生细胞,所述细胞在相同或相关生物学途径中的蛋白质复合物、RNA或蛋白质,彼此作为上下游的RNA或蛋白质,彼此具有调节、激活或抑制功能的RNA或蛋白质,依赖彼此功能或活性的RNA或蛋白质,同源(例如,序列、结构或功能同源)的RNA或蛋白质的表达提高。 For example, the method can be used to produce cells, the cells in the same or related biological pathway protein complexes, RNA or protein, RNA or protein as each other upstream and downstream, having mutually adjustable, activation or inhibition of RNA function or proteins, increased expression of another function or activity-dependent RNA or protein homology (e.g., sequences homologous structural or functional) RNA or protein. 例如,该方法可用于产生免疫球蛋白(例如,IgA、IgD、IgE、IgF和IgM)的轻链和重链或其抗原结合片段的表达提高的细胞系。 For example, the method can be used to produce immunoglobulins (e.g., IgA, IgD, IgE, IgF and IgM) light chain and heavy chain or antigen-binding fragment of an increased expression of cell lines. 所述免疫球蛋白可以是全人的、人源化的或嵌合的免疫球蛋白。 The immunoglobulin can be fully human, humanized or chimeric immunoglobulin. 转染入本发明的细胞的RNA可以包括编码感兴趣的蛋白质的RNA序列。 Transfected into cells of the present invention may comprise RNA RNA sequence encoding a protein of interest. 可按照本文所述方法生产任意蛋白质。 Any of the proteins may be produced according to the methods described herein. 可按照本发明所述方法生产的蛋白质的实例包括但不限于,肽激素(例如,胰岛素)、糖蛋白激素(例如,促红细胞生成素)、抗生素、细胞因子、酶、疫苗(例如,HIV疫苗,HPV疫苗、HBV疫苗)、抗癌治疗剂(例如,Mucl)和治疗性抗体。 Examples of proteins may be produced in accordance with the present invention include, but are not limited to, peptide hormones (e.g., insulin), glycoprotein hormones (e.g., erythropoietin), an antibiotic, cytokines, enzymes, vaccines (e.g., HIV vaccines , HPV vaccine, of HBV vaccine), anti-cancer therapeutic agents (e.g., of Mucl) and therapeutic antibodies. 在个别具体实施方式中,所述RNA编码免疫球蛋白或其抗原结合片段,例如免疫球蛋白的重链、免疫球蛋白的轻链、单链Fv、抗体片段(例如Fab、Fab'或(Fab' )2)或免疫球蛋白抗原结合片段。 In particular embodiments, the RNA encodes an immunoglobulin or antigen-binding fragment thereof, for example, a heavy chain, immunoglobulin light chain, single chain Fv, antibody fragment of an immunoglobulin (e.g. Fab, Fab 'or (Fab ') 2) or an immunoglobulin antigen-binding fragment. 在特定具体实施方式中,所述RNA编码促红细胞生成素。 In certain embodiments, the RNA encodes erythropoietin. 在另一特定具体实施方式中,所述RNA编码一个或多个结合细胞表面受体(表皮生长因子受体(EGFR)、HER2或c-ErbB-Ι)且任选地拮抗或激活细胞表面受体的免疫球蛋白或其片段,例如Erbitux™(cetuximab)。 In another particular embodiment, the RNA encoding one or more binding cell surface receptors (epidermal growth factor receptor (EGFR), HER2, or c-ErbB-Ι) and optionally activate or antagonize cell surface by immunoglobulins or fragments thereof, e.g. Erbitux ™ (cetuximab).

[0052] 蛋白质的分离或纯化 [0052] The separation or purification of proteins

[0053] 本文所述方法可以进一步包括分离或纯化通过本文所述方法制备的蛋白质、多肽或肽的步骤。 [0053] The methods described herein may further comprise the step of isolation or purification by the protein, polypeptide or peptide of the preparation methods described herein. 本领域普通技术人员可以容易地确定从培养的细胞中纯化或分离感兴趣的蛋白质的合适方式。 Those of ordinary skill in the art can readily determine the appropriate manner purified or isolated from cultured cells of interest in the protein. 通常,通过采用亲和结合的捕获方法或非亲和纯化而进行。 Typically, the method is carried out by using capturing or affinity purification of binding affinity. 如果感兴趣的蛋白质不被培养的细胞分泌,则在前述纯化或分离之前裂解培养的细胞。 If the protein of interest are not cultured cells secreted, purified or isolated before the lysis of the cells cultured. 可以使用包含感兴趣的蛋白质、细胞培养基成分和细胞培养添加成分(例如本发明中的消泡化合物和其他营养和补充物、细胞、细胞碎片、宿主细胞蛋白质、DNA、病毒等等)不明的细胞培养液体。 You may be used to add protein component of interest comprise, cell culture and cell culture media component (e.g. antifoam compounds of the present invention and other nutrients and supplements, cells, cell debris, host cell proteins, DNA, viruses and the like) unknown cell culture liquid. 此外,如果希望的话,该过程本身可以在生物反应器内进行。 Further, if desired, the process itself can be carried out in a bioreactor. 可以将所述液体预处理至期望的促进因素,例如pH、温度或其他促进因素,或者可以在加入聚合物后对液体进行处理,或者可以将聚合物加入到载液(carrier liquid)中,所述载液被正确处理至促进条件所需的参数,而所述促进条件是待溶解至液体中的聚合物所需的。 The liquid may be pretreated to promote the desired factors such as pH, temperature, or other factors to promote, or may be treated in the liquid after addition of the polymer or polymers may be added to the carrier liquid (carrier liquid) in the said carrier liquid is treated to promote proper parameters necessary condition, and the condition that the accelerator to the polymer to be dissolved in the liquid desired. 多聚物使得与所述液体完全流转,而后施加促进因素(改变pH、温度、盐浓度等等),目标蛋白质和多聚物从溶液中沉淀出来。 Polymer such that the liquid flow completely, and then applied to promoting factor (altered pH, temperature, salt concentration, etc.), the target protein and polymers precipitate out of solution. 将多聚物和目标蛋白质从液体剩余部分分离并任选地洗涤一次或多次以去除任何粘合的或松散结合的杂质。 The polymer and the protein isolated from the remainder of the liquid and optionally washed one or more times to remove any loosely bound or adhered impurities. 随后通过例如洗提等自多聚物回收所述目标蛋白质。 By subsequently eluting the like, for example, polymer recovered from the target protein. 一般,在一系列的条件下完成所述洗提,从而使聚合物保持其固体(沉淀)形式,并且在期望蛋白质的选择性洗提过程中保留其任意杂质。 In general, under a range of conditions to complete the elution, so that the polymer remains a solid (precipitated) form, and the desired selectivity of the process of elution of proteins retain their any impurities. 或者,可以将聚合物和蛋白质以及任意杂质溶解在新的液体(例如水或缓冲液)中,并且通过与多聚物或杂质相比,对蛋白质更具偏爱性和选择性的方式,例如亲和、离子交换、疏水或其他种类的层析方法回收所述蛋白质。 Alternatively, the polymer and the protein, and any impurity can be dissolved in the new liquid (e.g., water or a buffer solution), and by comparison with polymer or impurities, and the preference of the protein more selective manner, e.g. pro and, ion exchange, hydrophobic, or other types of chromatographic methods recovering the protein. 随后回收所洗提的蛋白质,并且如果需要对其进行额外的加工步骤,或者传统的分批式步骤或者连续流加步骤(如果合适)。 Subsequently recovering the protein eluted, and if necessary subjected to additional processing steps, or the step of the conventional batch or continuous flow plus step (if appropriate).

[0054] 此外,优化特定多肽在可能感兴趣的细胞系或细胞系集中的表达是有用的,尤其是工程化蛋白,例如具有已知活性的对照蛋白的蛋白变体。 [0054] Further, optimization of particular polypeptides in concentrated cell lines or cell lines expressing interest may be useful, especially engineered protein, e.g. with a known control protein activity protein variants. 在一种具体实施方式中,提供了通过提供多种靶细胞种类,并且使所述多种靶细胞种类的每一种均独立地与编码工程化多肽的修饰mRNA接触,而优化工程化蛋白质在靶细胞中的表达的方法。 In a specific embodiment, there is provided by providing a plurality of types of target cells, and the modified mRNA species in contact with said plurality of target cells each of which is independently encoding engineered polypeptides, protein engineering optimized the method of expression of a target cell. 此外,可以改变培养条件以提高蛋白质生产效率。 In addition, the culture conditions can be varied to improve the efficiency of protein production. 随后,检测和/或量化多种靶细胞类型中工程化多肽的存在和/或水平,使得可以通过选择有效的靶细胞和与之相关的细胞培养条件而对工程化多肽的表达进行优化。 Subsequently, to detect and / or quantify the presence of more target cell types engineered polypeptide and / or levels, can be optimized by selecting such target cells and effective expression related to cell culture conditions engineered polypeptide. 特别地,当工程化多肽包含一个或多个翻译后修饰或具有实质的三级结构或常使蛋白质高效生产复杂化的条件时,此类方法尤其有用。 In particular, when the engineered polypeptide comprises one or more post-translational modification, or tertiary structure, or have substantial conditions often make the efficient production of a protein complex of such methods is particularly useful.

[0055] “感兴趣的蛋白”或“目标蛋白”包括在本文提供的蛋白及其片段、突变体、变体及改变。 [0055] "protein of interest" or "target protein" includes proteins and fragments thereof provided herein, mutants, variants and alterations. 特别地,目标蛋白/多肽或感兴趣的蛋白的实例为,但不限于,胰岛素,胰岛素样生长因子,人生长激素(hGH),组织纤溶酶原激活剂(tPA),细胞因子,例如白介素(IL),例如IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、IL-16、IL-17、IL-18,干扰素(IFN) a , IFN β、IFN Y、IFNQ 或IFNt,肿瘤坏死因子(TNF),例如TNF α和TNF β > TNF y , TNF相关细胞凋亡诱导配体(TRAIL);粒细胞集落刺激因子(G-CSF),粒细胞-巨噬细胞集落刺激因子(GM-CSF),巨噬细胞集落刺激因子(M-CSF),单核细胞趋化蛋白-1 (MCP-1)和血管内皮生长因子(VEGF)。 In particular, protein / polypeptides or proteins of interest Examples are, but not limited to, insulin, insulin-like growth factor, human growth hormone (of hGH), tissue plasminogen activator (of tPA), cytokines, such as interleukin (IL), e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL -12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) a, IFN β, IFN Y, IFNQ or IFNt, tumor necrosis factor (TNF) such as TNF α and TNF β> TNF y, TNF-related apoptosis-inducing ligand (of TRAIL); granulocyte colony stimulating factor (G-CSF), granulocyte - macrophage colony stimulating factor (GM-CSF), giant macrophage colony stimulating factor (M-CSF), monocyte chemoattractant protein -1 (MCP-1) growth factor (VEGF) and vascular endothelium. 还包括促红细胞生成素或其他激素生长因子的生产。 Further comprising the production of erythropoietin or other hormone growth factors. 按照本发明的方法还可以有利地用于生产抗体或其片段。 The method according to the present invention can also be advantageously used to produce antibodies or fragments thereof. 所述片段包括,例如,Fab片段(抗原结合片段)。 The fragments include, for example, Fab fragments (antigen binding fragment). Fab片段是由通过相邻恒定区连在一起的双链可变区组成的,其可以通过蛋白酶(例如木瓜蛋白酶)消化常规抗体而形成,但是相似的Fab片段也同时会通过遗传工程产生。 Fab fragments are double-stranded variable region joined together by the adjacent constant region, which may be formed by protease (e.g., papain) digestion of conventional antibodies, but similar Fab fragments may also be produced by genetic engineering. 其他的抗体片段包括F(ab')2片段,可通过胃蛋白酶酶解制备。 Other antibody fragments include F (ab ') 2 fragments, it can be produced by pepsin digestion.

[0056] 一般而言,感兴趣的蛋白是作为分泌多肽自培养基中回收的,或者,如果没有分泌性信号表达,则可从宿主细胞裂解物中回收。 [0056] In general, the protein of interest from the culture medium as a secreted polypeptide recovered, or, if there is no expression of secretory signal may be recovered from host cell lysates. 需要采用可以获得基本上均一的感兴趣蛋白质制备物的方式自其他重组蛋白质和宿主细胞蛋白质中纯化感兴趣的蛋白质。 You need be obtained substantially homogeneous protein preparation of interest from other recombinant proteins manner and host cell proteins in the purified protein of interest. 作为第一步,自培养基或裂解物中移除细胞和/或粒状细胞碎片。 As a first step, removing cells and / or particulate cell debris from the culture medium or lysate. 随后,通过例如免疫亲合或离子交换柱分级、醇沉淀、反相HPLC、Sephadex色谱、硅石色谱或诸如DEAE的阳离子交换树脂色谱,自可溶性蛋白、多肽和核酸杂质中纯化感兴趣的产物。 Then, for example, by immunoaffinity or ion-exchange column fractionation, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or cation exchange resin such as DEAE chromatography, bought from soluble proteins, polypeptides and nucleic acids purified product impurities. 通常,教导本领域技术人员如何纯化宿主细胞表达的异源蛋白质的方法是本领域已知的。 The method is usually a heterologous protein, teach those skilled in the art how purified host cell expression are known in the art. 所述方法例如描述在(Harrisand ngal, Protein Purification Methods:A Practical Approach, Oxford UniversityPress,1995)或(Robert Scopes, Protein Purification:Principles and Practice,Springer, 1988)中。 For example, the method described in (Harrisand ngal, Protein Purification Methods: A Practical Approach, Oxford UniversityPress, 1995): In (Principles and Practice, Springer, 1988 Robert Scopes, Protein Purification), or.

[0057] 本发明的方法促进了核酸向体内、离体或培养物中细胞群的递送。 Method [0057] The nucleic acids of the present invention to facilitate the in vivo, ex vivo cell populations or cultures of delivery. 例如,将包含大量宿主细胞(例如,真核细胞,如酵母或哺乳动物细胞)的细胞培养物与含有增强的核酸的组合物接触,所述增强的核酸具有至少一个核苷修饰以及任选的可翻译区。 For example, a host cell containing a large amount (e.g., eukaryotic cells, such as yeast or mammalian cells) of the cell culture is contacted with a nucleic acid composition comprising enhanced, the enhancement of the nucleic acid having at least one modified nucleoside and optionally translatable region. 所述组合物通常还包含转染试剂或其他提高增强的核酸向宿主细胞的摄入效率的化合物。 Typically, the composition further comprising a compound uptake efficiency transfection reagent or other reinforcement to improve the nucleic acid into a host cell. 与相应未修饰核酸相比,所述增强的核酸在细胞群中显示出增强的保留能力。 Compared to a corresponding non-modified nucleic acid, said nucleic acid exhibits enhanced retention capacity increased in the cell population. 所述增强的核酸的保留能力比未修饰的核酸的保留能力高。 The enhanced ability to retain the nucleic acid retention capacity higher than unmodified nucleic acid. 在一些具体实施方式中,比未修饰核酸的保留能力至少高约50%、75%、90%、95%、100%、150%,200%或超过200%。 In some embodiments, about 50% than unmodified nucleic acid retention capacity is at least 75%, 90%, 95%, 100%, 150%, 200%, or more than 200%. 这样的保留能力优势可以通过使用增强的核酸进行一轮转染获得,或者可以通过后续多轮重复转染获得。 Such advantages retention capacity can be obtained by a round of transfection using enhanced nucleic acid, or may be obtained by subsequent repeated rounds of transfection.

[0058] 在一些具体实施方式中,增强的核酸与一个或多个其他核酸被递送到靶细胞群。 [0058] In some embodiments, the enhanced with one or more other nucleic acid nucleic acid is delivered to the target cell population. 这种递送可以同时进行,或者在递送一个或多个其他核酸之前递送增强的核酸。 Such delivery may be performed simultaneously, a nucleic acid delivery-enhancing or prior to delivery one or more other nucleic acids. 所述其他一个或多个核酸可以是修饰的核酸或未修饰的核酸。 The one or more additional nucleic acid can be modified or unmodified nucleic acids. 应该理解,起始存在的增强的核酸基本上不诱导细胞群的天然免疫反应,并且随后存在的未修饰的核酸也不会激活天然免疫反应。 It should be appreciated that the starting nucleic acid present in increased substantially does not induce the innate immune response of the cell population, and the unmodified nucleic acid is then present and does not activate the innate immune response. 因此,如果期望在靶细胞群中存在的蛋白质由未修饰的核酸翻译,则增强的核酸自身可以不含可翻译区。 Thus, if present in the target cell population by the unmodified nucleic acid protein translation, the desired nucleic acid may itself be enhanced free of untranslated region.

[0059] 拮抗蛋白的表达 Expression [0059] antagonistic protein

[0060] 本文所述方法和组合物可用于制备能削弱或阻止哺乳动物受试者的内源性激动生物反应和/或拮抗受体或信号转导分子的蛋白质。 [0060] The methods and compositions described herein can be used to prepare weakens or prevents endogenous inflammatory biological response of a mammalian subject and / or antagonizing receptor signal transduction molecules or proteins. 例如,IL-12和IL-23受体信号转导在慢性自体免疫病(例如多发性硬化)和炎性疾病(例如类风湿性关节炎、银屑病、红斑狼疮、强直性脊柱炎和克罗恩氏病)中增强(Kikly K, Liu L, Na S Sedgwick JD(2006)Curr.0pin.1mmunol.第18卷第6期,第670 - 5页)。 For example, IL-12 and IL-23 receptor signaling in chronic autoimmune diseases autologous (e.g., multiple sclerosis) and inflammatory diseases (e.g. rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis, and g Crohn's disease) enhanced (Kikly K, Liu L, Na S Sedgwick JD (2006) Curr.0pin.1mmunol Vol. 18 No. 6, 670 - page 5). 另一具体实施方式中,核酸编码针对趋化因子受体的拮抗剂。 In another specific embodiment, the nucleic acid encoding a chemokine receptor antagonist. 趋化因子受体CXCR-4和CCR-5是HIV进入宿主细胞所需的(Arenzana-Seisdedos F 等,(1996) Nature, 3 月10 日;第383 卷第6599 期,第400 页)。 Chemokine receptor CXCR-4 and CCR-5 is the HIV entry desired host cell (Arenzana-Seisdedos F, etc., (1996) Nature, 10 March; Vol. 383 6599, on page 387.).

[0061] 靶物质(moiety)。 [0061] The target substance (moiety). 本发明的具体实施方式中,提供修饰的核酸以表达蛋白质结合伴侣或位于细胞表面的的受体,所述受体的功能是在体内或体外将细胞祀向特定组织空间或者使其与特定物质反应。 DETAILED DESCRIPTION The present invention provides a modified nucleic acid, or to express a protein binding partner on the cell surface receptor, the receptor function in vivo or in vitro the cells to a specific tissue space or Si reacted with a particular substance reaction. 合适的蛋白质结合伴侣包括抗体及其功能片段、支架蛋白质或肽。 Suitable protein binding partners include antibodies and functional fragments, protein or peptide scaffold. 此外,修饰的核酸可用于指导脂类、糖类或其他生物物质的合成和细胞外定位。 In addition, modified nucleic acids may be used to guide lipids, carbohydrates or other synthetic extracellular targeting and biological substances.

[0062] 永久件基因表汰沉默。 [0062] permanently table member eliminating gene silencing. 在哺乳动物受试者中通过表观遗传学沉默基因表汰的方法,该方法包括核酸,所述核酸的可翻译区编码一种或多种多肽,所述多肽能够指导序列特异性组蛋白H3甲基化,从而起始异染色质的形成并且减少特定基因周围的基因转录,实现沉默基因的目的。 In a mammalian subject by methods apparent elimination genetic silencing of gene expression, the method comprising the nucleic acid, the nucleic acid may encode one or more untranslated region polypeptide sequence capable of directing specific histone H3 methyl, thereby starting heterochromatin formation and reduce transcription of a gene around the particular gene, the gene silencing to achieve the purpose. 例如,酪氨酸激酶2基因的功能获得性突变是骨髓增生疾病家族的原因。 For example, tyrosine kinase 2 gene function mutations myeloproliferative disease is the cause of the family.

[0063] 机理详情。 [0063] mechanism details. 裂殖酵母需要2个RNAi复合体用于siRNA介导的异染色质装配:RNA诱导的转录沉默(RITS)复合体和RNA引导的RNA聚合酶复合体(RDRC) (Motamedi等,Cell,2004年,119,第789-802页)。 Fission yeast requires a complex two RNAi siRNA mediated heterochromatin assembly: RNA-induced transcriptional silencing (the RITS) complexes and RNA directed RNA polymerase complex (RDRC) (Motamedi et, Cell, 2004 119, pp. 789-802). 裂殖酵母中,RITX复合体包括siRNA结合Argonaute家族蛋白Agol,染色质结构域蛋白质Chpl和Tas3。 S. pombe, RITX complex comprises siRNA binding Argonaute family proteins Agol, chromatin domain protein Chpl and Tas3. 裂殖酵母RDRC复合体由RNA依赖性RNA聚合酶Rdpl、推定的RNA螺旋酶Hrrl和polyA聚合酶家族蛋白Cidl2组成。 Fission yeast RDRC complex by an RNA-dependent RNA polymerase Rdpl, putative polyA RNA polymerase and a helicase family protein Hrrl Cidl2 composition. 这两种复合物需要Dicer核糖核酸酶和Clr4组蛋白H3甲基转移酶才能有活性。 Both composite and Clr4 of Dicer ribonuclease histone H3 methyltransferase activity can have. 同时,Agol与siRNA分子(Dicer介导的对Rdpl共转录产生的dsRNA转录本进行切割而产生)结合并且允许Chpl、Tas3、Hrrl和Clr4与DNA区域序列特异性直接结合,所述DNA区域必定用于甲基化和组蛋白修饰并随后包装成转录水平沉默的异染色质。 Meanwhile, the siRNA molecule Agol (dsRNA Dicer transcription mediated transcription co-present Rdpl cut generated) and allowed to bind Chpl, Tas3, Hrrl Clr4 and direct binding sequence-specific DNA region, said DNA region bound by in methylation and histone modification, and then packaged as transcriptional silencing heterochromatin. 虽然该机制与DNA着丝粒区域顺式发挥作用,但通过针对DNA特定区域的双链siRNA的共转染和杂RNAi引导的siRNA核糖核酸酶Eril沉默使序列特异性反式沉默成为可能(Buhler等.Cell2006,125,873-886)。 The mechanism of the DNA and the role of cis-centromeric regions, but by targeting specific regions of DNA double strand siRNA in cotransfection and heteroaryl directed RNAi silencing siRNA ribonuclease Eril sequence-specific silencing trans possible (a Buhler etc. .Cell2006,125,873-886). [0064] 制备多肽变体 [0064] Preparation of polypeptide variants

[0065] 本文所述方法和组合物可用于制备多肽变体。 [0065] The methods and compositions described herein may be used for preparing polypeptide variants. 本文提供了编码与对照多肽序列具有一定同一性的变体多肽的核酸。 This article provides a nucleic acid variant encodes a polypeptide sequence with the control having a certain identity to the polypeptide. 术语“同一性”如本领域所知,是指通过比较序列而测定的两条或更多多肽序列之间的相互关系。 The term "identity" as known in the art, refers to the relationship between one or more polypeptide sequences as determined by comparing two sequences. 在本领域,“同一性”还表示通过两条或更多氨基酸残基链之间的匹配数量而测定的肽之间的序列相关性。 In the art, "identity" also means the correlation between the peptide sequence determined by the number of matches between the two or more amino acid residues chain. “同一性”测定两条或更多序列的较小者之间相同匹配的百分数,其以特定的数学模型或计算机程序(例如,“运算法则”)解决缺口序列(若有的话)。 "Identity" Determination of the percentage of identical matches between the smaller of two or more sequences that addresses a particular sequence gaps mathematical model or computer program (e.g., "algorithms") (if any). 通过已知方法可以容易地计算相关肽的同一性。 Identity of related peptides can be readily calculated by known methods. 这样的方法包括,但不限于,Computational Molecular Biology, Lesk, A.Μ., ed., Oxford University Press, New York, 1988;Biocomputing:1nformatics andGenome Projects, Smith, D.ff., ed., Academic Press, New York, 1993;Computer Analysisof Sequence Data, Parti, Griffin, AM, and Griffin, HG, eds., Humana Press, NewJersey,1994;Sequence Analysis in Molecular Biology,von Heinje, G.,AcademicPress, 1987;Seq uence Analysis Primer, Gribskov, M.and Devereux, J., eds., M.StocktonPress, New York, 1991;and Carillo et al., SIAM J.Applied Math.48, 1073(1988)。 Such methods include, but are not limited to, Computational Molecular Biology, Lesk, A.Μ., ed, Oxford University Press, New York, 1988; Biocomputing:. 1nformatics andGenome Projects, Smith, D.ff., ed, Academic Press. , New York, 1993; Computer Analysisof Sequence Data, Parti, Griffin, AM, and Griffin, HG, eds, Humana Press, NewJersey, 1994;. Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Seq uence Analysis Primer, Gribskov, M.and Devereux, J., eds, M.StocktonPress, New York, 1991;. and Carillo et al, SIAM J.Applied Math.48, 1073 (1988)..

[0066] 在一些具体实施方式中,多肽变体与对照多肽具有相同或相似活性。 [0066] In some embodiments, the control polypeptide variant polypeptides have the same or similar activity. 或者,变体具有相对于对照多肽的改变的活性(例如,增加或降低)。 Alternatively, the variants have altered activity relative to the control polypeptides (e.g., increase or decrease). 通常,当通过本文所述的和本领域技术人员已知的序列比对程序以及参数测定时,本发明的特定多核苷酸或多肽的变体与特定对照多核苷酸或多肽相比,具有至少大约40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或更高的序列同一性。 Typically, and as described herein by those skilled in the known sequence alignment programs and parameters of the measurement, the present invention is a particular polynucleotide or polypeptide variant with a particular polynucleotide or polypeptide compared to a control, having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more sequence identity.

[0067] 如本领域技术人员所知,蛋白质片段、功能性蛋白质结构域和同源蛋白质也被认为是落入本发明的范围。 [0067] As those skilled in the art, protein fragments, and functional protein domains homologous proteins are also considered to fall within the scope of the invention. 例如,本文提供了对照蛋白质的任意蛋白质片段(表示比对照多肽序列短至少一个氨基酸残基的多肽序列,但其他部分相同),其具有大约10、20、30、40、50、60、70、80、90、100或多于100个氨基酸长度。 For example, any of the proteins provided herein to control protein fragments (expressed polypeptide sequence shorter than the control polypeptide sequence of at least one amino acid residue, but other portions of the same), which has about 10,20,30,40,50,60,70, 80, 90 or more than 100 amino acids in length. 另一个实施例中,可以依照本发明使用具有约20、约30、约40、约50或约100个氨基酸的任何蛋白质,其与任一本文所述序列相比,具有约40%、约50%、约60%、约70%、约80%、约90%、约95%或约100%的同一性。 Another embodiment, may have a use in accordance with the present invention, about 20, about 30, about 40, about 50, or any protein of about 100 amino acids, as compared with any one of the sequences described herein, having about 40%, about 50 %, about 60%, about 70%, about 80%, about 90%, about 95% or about 100% identity. 在某些具体实施方式中,如本文提供或涉及的任一序列中所示,按照本发明而使用的蛋白质序列包括 In certain embodiments, provided herein or as shown in any of the sequences involved in the protein sequence according to the present invention comprises the use of

2、3、4、5、6、7、8、9、10 或更多突变。 8, 9 or more mutations.

[0068] 制备多肽文库 [0068] Preparation of libraries of polypeptides

[0069] 本文所述方法和组合物可用于多肽文库的构建。 [0069] The methods and compositions described herein can be used to construct a library of polypeptides. 本文提供包含核苷修饰的多核苷酸文库,其中所述多核苷酸各自包含编码多肽(例如抗体、蛋白质结合伴侣、支架蛋白和本领域已知的其他多肽)的第一核酸序列。 This article provides a polynucleotide library comprising a modified nucleoside, wherein the polynucleotide sequences each comprising a nucleic acid encoding a first polypeptide (e.g. an antibody, a protein binding partner, scaffold proteins and other polypeptides known in the art) is. 一般而言,多核苷酸是适于直接引入目标宿主细胞形式的mRNA,所述目标宿主细胞合成所编码的多肽。 Generally, the polynucleotide is introduced directly into the target host cell is adapted to the form of the mRNA, the target host cells synthesize the encoded polypeptide.

[0070] 在某些具体实施方式中,制备蛋白质的多种变体(每一个均具有不同的氨基酸修饰)并测试以确定在药物代谢动力学、稳定性、生物适应性和/或生物活性或生物物理学特性(例如表达水平)方面的最佳变体。 [0070] In certain embodiments, several variations of a protein preparation (each having a different amino acid modifications) and tested to determine the kinetics of drug metabolism, stability, biocompatibility and / or biological activity, or biophysical properties (e.g. expression levels) optimal variant aspect. 这样的文库可以包含约10、102、103、104、105、106、107、IO8UO9或超过IO9种可能的变体(包括替换、删除一个或多个残基,和插入一个或多个 Such libraries may comprise from about 10,102,103,104,105,106,107, IO8UO9 or more than IO9 possible variations (including substitutions, deletions of one or more residues, and insertion of one or more

残基)。 Residues).

[0071 ] 制备多肽-核酸复合物 [0071] Preparation of polypeptide - nucleic acid complex

[0072] 本文所述方法和组合物可用于制备多肽-核酸复合物。 [0072] The methods and compositions described herein can be used to prepare polypeptide - nucleic acid complex. 合适的蛋白质翻译包括与mRNA相关的多个多肽和核酸的物理聚集。 Suitable proteins include physical aggregation translation of mRNA associated with a plurality of polypeptides and nucleic acids. 本发明提供蛋白质-核酸复合物,包含具有一个或多个核苷修饰(例如,至少2个不同的核苷修饰)的可翻译mRNA以及与mRNA结合的一个或多个多肽。 The present invention provides a protein - nucleic acid complex, comprising a translatable mRNA having one or more modified nucleosides (e.g., at least two different nucleoside modifications) and one or more polypeptides bound to the mRNA. 通常地,按有效阻止或减少所述复合物所引入的细胞的天然免疫反应的量提供蛋白质。 Generally, in an effective amount to prevent or reduce the innate immune response of the composite provided the introduced cellular proteins.

[0073] 制备不可翻译的修饰核酸 [0073] Preparation of modified nucleic acid untranslatable

[0074] 本文所述方法和组合物可用于制备不可翻译的修饰核酸。 [0074] The methods and compositions described herein can be used in the preparation of modified nucleic acid untranslatable. 如本文所述,提供具有基本上不可翻译的序列的mRNA。 As described herein, there is provided a substantially non-translatable mRNA having the sequence of. 当施用于哺乳动物受试者时,所述mRNA是有效的疫苗。 When administered to a mammalian subject, said mRNA is an effective vaccine.

[0075] 本文还提供了包含一个或多个非编码区的修饰核酸。 [0075] Also provided herein are modified nucleic acid comprising one or more non-coding regions. 这种修饰核酸通常不翻译,但是能结合和螯合一个或多个翻译器组分,例如核糖体蛋白或转移RNA(tRNA),从而有效减少细胞内的蛋白质表达。 Such modified nucleic acids are often not translated, and capable of binding to one or more translators chelating components, such as ribosomal protein or transfer RNA (tRNA), thus effectively reducing the expression of proteins within the cell. 该修饰核酸可以包含小核仁RNA(sno-RNA)、微小RNA(miRNA)、小干扰RNA(siRNA)、小发卡RNA(shRNA)或Piw1-相互作用RNA(piRNA)。 The modified nucleic acid may contain small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) or Piw1- interacting RNA (piRNA).

[0076] 修饰核酸 [0076] The modified nucleic acid

[0077] 本发明提供利了用含有一个或多个修饰核苷的包括RNA (例如信使RNA (mRNA))在内的核酸(称为“修饰的核酸(或修饰核酸)”)生产蛋白质的方法,所述修饰核酸具有有用的特性,包括基本上缺失诱导该mRNA所引入的细胞的天然免疫反应。 [0077] The present invention provides an advantage of containing one or more modified nucleosides of nucleic acids include RNA (e.g., messenger RNA (the mRNA)) including (referred to as "modified nucleic acid (or modified nucleic acid)") a method for producing a protein the modified nucleic acid having useful properties, including deletion of substantially the immune response induced by the native mRNA introduced cells. 由于这些修饰核酸增强了蛋白质的生产效率、核酸的细胞内保留能力和所接触的细胞的生存能力,并且具有降低的免疫原性,具有这些性质的核酸在本文中称为“增强的核酸(或增强核酸)”。 Since these modified nucleic acid to enhance productivity of protein, and the ability to retain the viability of cells in contact with the cells a nucleic acid, and has reduced immunogenicity, nucleic acids having these properties is referred to as "Enhanced nucleic acid (herein, or enhance the nucleic acid). "

[0078] 术语“核酸”,以其最广泛的含义包括引入或者能够引入至寡核苷酸链的任意化合物和/或物质。 [0078] The term "nucleic acid", in its broadest sense to include the introduction of any compound can be introduced or oligonucleotide chain and / or substances. 可用于本发明的示例性核酸包括,但不限于本文详细描述的一个或多个DNA、包括信使mRNA (mRNA)在内的RNA或DNA与RNA的杂合体、RNA干扰(RNAi)诱导剂、RNAi 药剂、小干扰RNA(siRNAs)、小发卡RNA(shRNAs)、微小RNA(miRNAs)、反义RNA、核酶、催化性DNA、诱导三螺旋形成的RNA、适体、载体等。 Exemplary nucleic acids can be used in the present invention include, but are not limited to one or more of the DNA described in detail herein, including messenger mRNA (mRNA) RNA or a hybrid of DNA and RNA, RNA interference (RNAi) inducing agents, RNAi agent, small interfering RNA (the siRNAs) and small hairpin RNA (of shRNAs), micro RNA (of miRNAs), antisense RNA, ribozymes, catalytic the DNA, RNA induce triple helix formation, aptamer, carriers and the like.

[0079] 本文提供了含有可翻译区和一个、二个或多于二个不同的核苷修饰的修饰核酸。 [0079] Provided herein and translated region containing one, two or more than two different nucleoside modifications modified nucleic acid. 在一些具体实施方式中,与相应的未修饰核酸相比,修饰核酸在该核酸所引入的细胞中显示出减少的降解。 In some embodiments, compared to the corresponding unmodified nucleic acids, modified nucleic acids show reduced degradation in the cell in which the nucleic acid is introduced. 例如,与相应的未修饰核酸的降解率相比,修饰核酸的降解率降低了大约10%、20%、30%、40%、50%、60%、70%、80%、90%或多于90%。 For example, the degradation rate compared to the corresponding unmodified nucleic acid, modified nucleic acid degradation rate is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more to 90%. 示例性的核酸包括核糖核酸(RNA)、脱氧核糖核酸(DNA)、苏糖核酸(TNA)、乙二醇核酸(GNA)或其杂合体。 Exemplary nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA) or hybrids thereof. 在典型的具体实施方式中,修饰核酸包括信使RNA (mRNA)。 In an exemplary embodiment, the modified nucleic acid comprising messenger RNA (mRNA). 如本文所述,本发明的核酸基本不诱导该mRNA所引入的细胞的天然免疫反应。 As described herein, the nucleic acid of the present invention substantially does not induce an immune response to the native mRNA introduced cells.

[0080] 在一些具体实施方式中,修饰核苷包括喃唳-4-酮核苷、5-氮-尿苷、2-硫-5-氮-尿苷、2-硫尿苷、4-硫-假尿苷、2-硫-假尿苷、5-羟基尿苷、3-甲基尿苷、5-羧甲基-尿苷、1-羧甲基-假尿苷、5-炔丙基-尿苷、1-炔丙基-假尿苷、5-牛磺酸甲基尿苷、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫-尿苷、1-牛磺酸甲基-4-硫-尿苷、5-甲基-尿苷、1-甲基-假尿苷、4-硫-1-甲基-假尿苷、2-硫-1-甲基-假尿苷、1-甲基_1_去氮_假尿苷、2-硫-1-甲基-1-去氮_假尿苷、二氢尿苷、二氢假尿苷、2_硫-二氢尿苷、2-硫-二氢假尿苷、2-甲氧基尿苷、2-甲氧基-4-硫-尿苷、4-甲氧基-假尿苷和4-甲氧基-2-硫-假尿苷。 [0080] In some embodiments, the modified nucleosides include Li thiopyran-4-one nucleosides, 5-N - uridine, 2-thio -5- N ​​- uridine, uridine 2-thiouracil, 4-thiouracil - pseudouridine, 2-thio - pseudouridine, 5-hydroxy-uridine, 3-methyl-uridine, 5-carboxymethyl - uridine, 1-carboxymethyl - pseudouridine, 5-propargyl- - uridine, 1-propargyl - pseudouridine, 5-methyluridine taurine, methyl taurine 1- - pseudouridine, 5-methyl-2-taurine sulfur - uridine , l-methyl-4-taurine sulfur - uridine, 5-methyl - uridine, 1-methyl - pseudouridine, 4-thiouracil-1-methyl - pseudouridine, 2-thio - 1-methyl - pseudouridine, 1-methyl-deaza _1_ _ pseudouridine, 2-methyl-1-deaza sulfur _ pseudouridine, dihydrouridine, urinary dihydro false glycosides, sulfur 2_ - dihydro-uridine, 2-thio - dihydro pseudouridine, 2-methoxy-uridine, 2-methoxy-4-sulfur - uridine, 4-methoxy - false Urine 4-methoxy-glycosides and sulfur - pseudouridine.

[0081] 在一些具体实施方式中,修饰核苷包括5-氮-胞苷、伪异胞苷、3-甲基胞苷、N4-乙酰基胞苷、5-甲酰基胞苷、N4-甲基胞苷、5-羟甲基胞苷、1-甲基-伪异胞苷、吡咯并胞苷、吡咯并伪异胞苷、2-硫-胞苷、2-硫-5-甲基-胞苷、4-硫-伪异胞苷、4-硫-1-甲基-伪异胞苷、4-硫-1-甲基-1-去氮-伪异胞苷、1-甲基-1-去氮-伪异胞苷、zebularine、5_ 氮-zebularine、5_ 甲基-zebularine、5_ 氮-2- 硫-zebularine、2-硫-Zebularine、2-甲氧基-胞苷、2-甲氧基-5-甲基-胞苷、4-甲氧基-伪异胞苷和4-甲氧基-1-甲基-伪异胞苷。 [0081] In some embodiments, the modified nucleoside include 5-N - cytidine, pseudo-isocytidine, 3-methyl cytidine, N4-acetyl cytidine, 5-formyl-cytidine, N4-A cytidine-yl, 5-hydroxymethyl cytidine, 1-methyl - pseudo iso cytidine, cytidine pyrrolo, pyrrolo pseudo-isocytidine, 2-thio - cytidine, 5-methyl-2-thio - cytidine, 4-thiouracil - pseudo-isocytidine, 4-thiouracil-1-methyl - pseudo-isocytidine, 4-methyl-1-deaza sulfur - pseudo-isocytidine, 1-methyl - 1- deaza - pseudo-isocytidine, zebularine, 5_ nitrogen -zebularine, 5_ methyl -zebularine, 5_ nitrogen -2- sulfur -zebularine, 2- sulfur -Zebularine, 2- methoxy - cytidine, 2- methyl-5 - cytidine, 4-methoxy - pseudo-isocytidine and 4-methoxy-1-methyl - pseudo-isocytidine.

[0082] 在其他具体实施方式中,修饰核苷包括2-氨基嘌呤、2,6- 二氨基嘌呤、7-去氮-腺嘌呤、7-去氮-8-氮-腺嘌呤、7-去氮-2-氨基嘌呤、7-去氮-8-氮-2-氨基嘌呤、7-去氮-2,6-二氨基嘌呤、7-去氮-8-氮-2,6-二氨基嘌呤、1-甲基腺苷、N6-甲基腺苷、N6-异戊烯基腺苷、N6-(顺式羟基异戊烯基)腺苷、2-甲硫基-N6-(顺式羟基异戊烯基)腺苷、N6-甘氨酰氨基甲酰腺苷、N6-苏氨酰氨基甲酰腺苷、2-甲硫基-N6-苏氨酰氨基甲酰腺苷、N6, N6- 二甲基腺苷、7-甲基腺嘌呤、2-甲硫基-腺嘌呤和2-甲氧基-腺嘌呤。 [0082] In other embodiments, the modified nucleoside comprising a 2-aminopurine, 2,6-diaminopurine, 7-deaza - adenine, 7-deaza-aza - adenine, 7-deaza N-2-amino purine, 7- deaza-aza-2-amino purine, 7-deaza-2,6-diaminopurine, 7-deaza-aza-2,6-diaminopurine , 1-methyl adenosine, N6-methyl adenosine, N6-isopentenyl adenosine, N6-(cis-hydroxy-isopentenyl) adenosine, 2-methylthio--N6- (cis-hydroxy isopentenyl) adenosine, N6-glycylamino carboxamido adenosine, N6-threonyl acylamino carboxamido adenosine, 2-methylthio-threonyl -N6- acylamino carboxamido adenosine, N6, N6 - dimethyl-adenosine, 7-methyl-adenine, 2-methylthio-- adenine and 2-methoxy - adenine.

[0083] 在特定具体实施方式中,修饰核苷是5' -0-(1-硫代磷酸)_腺苷、5' -0-(1-硫代磷酸)_胞苷、5'-0-(1-硫代磷酸)-鸟苷、5'-0-(1-硫代磷酸)-尿苷或5'-0-(1-硫代磷 [0083] In certain embodiments, the modified nucleoside is a 5 '-0- (l phosphorothioate) _ adenosine 5' -0- (l phosphorothioate) _ cytidine, 5'-0 - (1-phosphorothioate) - guanosine 5'-O- (1-thio-phosphate) - or uridine 5'-O- (1-thio-P

酸)_假尿苷。 Acid) _ Pseudouridine.

[0084] [0084]

Figure CN103429606AD00161

[0085] 5' -O- (1-硫代磷酸)-腺苷 [0085] 5 '-O- (1- phosphorothioate) - adenosine

[0086] [0086]

Figure CN103429606AD00162

[0087] 5' -0-(1-硫代磷酸)-胞苷 [0087] 5 '-0- (l phosphorothioate) - cytidine

[0088] [0088]

Figure CN103429606AD00163

[0089] 5' -0-(1-硫代磷酸)-鸟苷 [0089] 5 '-0- (l phosphorothioate) - guanosine

[0090] [0090]

Figure CN103429606AD00171

[0091] 5' -0-(1-硫代磷酸)-尿苷 [0091] 5 '-0- (l phosphorothioate) - uridine

Figure CN103429606AD00172

[0093] 5' -0-(1-硫代磷酸)-假尿苷 [0093] 5 '-0- (l phosphorothioate) - pseudouridine

[0094] 本文提供α -硫取代的磷酸分子以通过非天然硫代磷酸酯骨架连接而赋予RNA和DNA聚合体以稳定性。 [0094] Provided herein are α - thio-substituted phosphate molecules through an unnatural backbone linkages and phosphorothioate RNA and DNA to impart stability to the polymer. 硫代磷酸DNA和RNA具有提高的核酸酶抗性并因此在细胞环境中具有更长的半衰期。 Phosphorothioate DNA and RNA having increased nuclease resistance and thus has a longer half-life in a cellular environment. 还预期硫代磷酸酯连接的核酸通过较弱地结合/激活细胞天然免疫分子而降低天然免疫反应。 Phosphorothioate linkages are also contemplated nucleic acid is lowered by the natural immune response to the weakly binding / activation of innate immune cell molecules.

[0095] 在某些具体实施方式中,例如,如果需要精确的蛋白质生产时间,期望在细胞内降解弓I入该细胞的修饰核酸。 [0095] In certain embodiments, for example, if protein production requires precise time, within the desired intracellular degradation bow I modified nucleic acid into the cell. 因此,本发明提供包含降解结构域的修饰核酸,在细胞内能够以被引导的方式作用于其上。 Accordingly, the present invention provides a nucleic acid modifying domain comprises a degradable, it can act thereon in a guided manner in a cell.

[0096] 在其他具体实施方式中,修饰核苷包括肌苷、1-甲基-肌苷、丫苷、怀丁苷(wybutosine)、7_去氣_鸟昔、7_去氣-8-氣-鸟昔、6_硫-鸟昔、6_硫-7-去氣_鸟昔、6-硫-7-去氮-8-氮-鸟苷、7-甲基-鸟苷、6-硫-7-甲基-鸟苷、7-甲基肌苷、6-甲氧基-鸟苷、1-甲基鸟苷、N2-甲基鸟苷、N2,N2-二甲基鸟苷、8-氧-鸟苷、7-甲基-8-氧-鸟昔、1_甲基_6-硫_鸟昔、N2-甲基_6-硫_鸟昔和N2, N2- _二甲基_6-硫_鸟昔。 [0096] In other embodiments, the modified nucleosides include inosine, 1-methyl - inosine, Ah glycosides, glycoside Whiting (wybutosine), 7_ _ degassing birds past, 7_ degassing -8- gas - birds past, 6_ sulfur - birds past, 6_ sulfur degassing _ -7- birds past, 6-aza deaza sulfur -7- - guanosine, 7-methyl - guanosine, 6- sulfur-7-methyl - guanosine, inosine 7-methyl, 6-methoxy - guanosine, 1-methyl guanosine, N2- methyl guanosine, N2, N2- dimethyl-guanosine, 8-oxo - guanosine, 7-methyl-8-oxo - Xi birds, birds _ 1_ methyl _6- Xi sulfur, sulfur N2- methyl _6- _ birds celecoxib and N2, N2- _ dimethyl _ sulfur group _6- birds past.

[0097] 核酸的其他组分是任选的,并且在一些具体实施方式中是有益的。 [0097] Other optional components are nucleic acid, and is advantageous in some embodiments. 例如,提供了5'非翻译区(UTR)和/或3'UTR,其中之一或两者能独立地包含一个或多个不同的核苷修饰。 For example, a 5 'untranslated region (UTR) and / or 3'UTR, wherein one or both independently contain one or more different nucleoside modifications. 在这样的具体实施方式中,核苷修饰也可以存在于可翻译区。 In such an embodiment, the modified nucleosides may also be present in the translation region. 还提供了具有Kozak序列的核酸。 Nucleic acid having a Kozak sequence.

[0098] 此外,还提供了具有一个或多个能从所述核酸剪切的内含子核苷酸序列的核酸。 [0098] In addition, a nucleic acid is also provided having one or more nucleic acids from said shear intron nucleotide sequence.

[0099] 进一步的,还提供了含有内核糖体进入位点(IRES)的核酸。 [0099] Further, also provides a nucleic acid containing the internal ribosome entry site (IRES) of. IRES可以作为单一核糖体结合位点起作用或也可以作为mRNA的多个核糖体结合位点之一而发挥功效。 IRES as a single ribosome binding site works or can be used as multiple mRNA ribosome binding sites and one to be effective. 包含多于一个功能性核糖体结合位点的mRNA可编码数个通过核糖体而独立翻译的肽或多肽(多顺反子mRNA)。 Comprising more than one binding site of the mRNA may encode a functional ribosome several independent translation by ribosomes peptide or polypeptide (polycistronic mRNA). 当提供具有IRES的核酸时,进一步任选提供第二可翻译区。 When providing a nucleic acid having an IRES, further optionally providing a second translatable region. 可用于本发明的IRES序列的实例包括但不限于,来自细小核糖核酸病毒(例如,FMDV)、鼠疫病毒(CFFV)、脊髓灰质炎病毒(PV)、脑心肌炎病毒(ECMV)、手足口病毒(FMDV)、丙肝病毒(HCV)、典型猪瘟病毒(CSFV)、鼠白血病病毒(MLV)、猴免疫缺陷病毒(SIV)或蟋蟀麻痹病毒(CrPV)。 Examples of IRES sequences used in the present invention include, but are not limited to, from a picornavirus (e.g., of FMDV), plague viruses (CFFv), poliovirus (the PV), encephalomyocarditis virus (the ECMV), foot and mouth virus ( FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV) or cricket paralysis virus (CrPV). _0] 利用修饰核酸阻止或降低天然细胞免疫反应的活化。 _0] using a modified nucleic acid prevents or reduces activation of natural cellular immune response. [0101] 本文所述的修饰核酸能够逃脱该核酸所引入的细胞的天然免疫反应,从而提高细胞内蛋白质的生产效率。 [0101] The modified nucleic acid described herein can escape the natural immune response of a nucleic acid is introduced into a cell, thereby improving the production efficiency of intracellular proteins. 术语“天然免疫反应”包括针对通常为病毒或细菌来源的外源单链核酸的细胞反应,其包括诱导细胞因子(特别是干扰素)表达和释放以及细胞死亡。 The term "innate immune response" comprises a generally cellular response against single stranded nucleic acid is an exogenous viral or bacterial origin, including induction of cytokine (particularly IFN) expression and release and cell death. 在天然细胞免疫反应中,蛋白质合成也降低。 Natural cellular immune response, the protein synthesis is reduced. 虽然消除细胞内天然免疫反应是有利的,但本发明提供了显著降低免疫反应(包括干扰素信号转导)但不完全消除该反应的修饰mRNA。 While the innate immune response is advantageous to eliminate the cells, the present invention provides a significant reduction in the immune response (including IFN signal transduction) but not completely eliminate the modified mRNA of the reaction. 在一些具体实施方式中,与由相应的未修饰核酸诱导的免疫反应相比,所述免疫反应降低大约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、99%、99.9% 或大于99.9%。 In some embodiments, as compared with the corresponding unmodified nucleic acid-induced immune response, the immune response is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95%, 99%, 99.9%, or greater than 99.9%. 这样的减少可以通过I型干扰素的表达或活性水平或者干扰素调节基因(例如Toll样受体,例如TLR7和TLR8)的表达而测量。 Such reduction may be either through IFN type I interferons regulate gene expression or activity level (e.g. Toll-like receptors, e.g. a TLR8 and TLR7) expressed measured. 天然免疫反应的减少也可以通过在向细胞群一次或多次施以修饰RNA之后细胞死亡的减少而测定;例如比用相应的未修饰核酸观察到的细胞死亡频率低约10%、25%、50%、75%、85%、90%、95%或超过95%。 Reducing the natural immune response can also be measured in one or more cell population subjected to modifications to reduce cell death after RNA; e.g. compared with the corresponding unmodified nucleic acid low-frequency cell death was observed about 10%, 25%, 50%, 75%, 85%, 90%, 95%, or more than 95%. 而且,细胞死亡可影响少于约50%、40%、30%、20%、10%、5%、1%、0.1%、0.01%或少于0.01%的与修饰核酸接触的细胞。 Moreover, cell death may affect less than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, or less than 0.01% of the cells in contact with the modified nucleic acid.

[0102] 本发明提供了重复地将修饰核酸引入(例如转染)至靶细胞群,例如在体外、离体或在体内。 [0102] The present invention provides a modified nucleic acid is introduced repeatedly (e.g., transfected) into a target cell population, for example, in vitro, ex vivo or in vivo. 接触细胞群的步骤可以重复一次或多次(例如,二次、三次、四次、五次或多于五次)。 The step of contacting the cell population may be repeated one or more times (e.g., two, three, four, five or more than five times). 在一些具体实施方式中,用修饰核酸接触细胞群的步骤重复多次,使其足以在细胞群内实现预定的蛋白质翻译效率。 In some embodiments, the step of contacting the cell population with the modified nucleic repeated many times, it is sufficient to achieve a predetermined efficiency of protein translation in a cell population. 考虑到由核酸修饰产生的降低的靶细胞群细胞毒性,在不同的细胞类型中都可实现所述重复转染。 Taking into account the reduced cytotoxicity of the target cell population produced by the modified nucleic acids, can be repeated to achieve the transfected in different cell types.

[0103] 修饰核酸的合成 [0103] Synthesis of modified nucleic acid

[0104] 用于本发明的核酸可以按照任何可行的技术制备,所述技术包括,但不限于化学合成,酶法合成(其通常是指体外转录),长前体的酶法或化学裂解等等。 [0104] A nucleic acid of the present invention can be prepared according to any feasible technique, the technique including, but not limited to chemical synthesis, enzymatic synthesis (which usually refers to in vitro transcription), long precursor enzymatic or chemical cleavage, etc. Wait. 合成RNA的方法在本领域已知(参见,例如,Gait, MJ(编)Oligonucleotide synthesis:a practicalapproach, Oxford[Oxfordshire], Washington, DC:1RL Press,1984 ;和Herdewijn,P.(1¾)Oligonucleotide synthesis:methods and applications,Methods in MolecularBiology, v.288 (Clifton, NJ) Totowa, NJ:Humana Press, 2005 ;两者通过引证而全文引入)。 RNA synthesis method known in the art (see, e.g., Gait, MJ (ed) Oligonucleotide synthesis: a practicalapproach, Oxford [Oxfordshire], Washington, DC: 1RL Press, 1984; and Herdewijn, P (1¾) Oligonucleotide synthesis. : methods and applications, Methods in MolecularBiology, v.288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; and both incorporated by reference).

[0105] 修饰核酸不需要沿该分`子的全长进行均一修饰。 [0105] The modified nucleic acid need not be uniform along the entire length of the sub-modified 'promoter. 不同的核苷酸修饰和/或骨架结构可以存在于核酸的不同位置。 And / or different positions may be present in the backbone structure of nucleic acids of different nucleotide modifications. 本领域普通技术人员理解,核苷酸类似物或其他修饰可以存在于核酸的基本不降低核酸功能的任意位置。 One of ordinary skill in the art understand, nucleotide analogs, or other modifications may be present in any position of the nucleic acid function is substantially reduced nucleic acid. 修饰可以是5'或3'末端修饰。 Modifications may be 5 'or 3' terminal modification. 核酸可以包括最少一个到最多100%修饰核苷酸,或任何中间的百分比,例如至少约50%修饰核苷酸、至少约80%修饰核苷酸或至少约90%修饰核苷酸。 Nucleic acid can include a minimum to a maximum of 100% modified nucleotides, or any intermediate percentage, eg, at least about 50% modified nucleotides, at least about 80% modified nucleotides or at least about 90% modified nucleotides.

[0106] 通常地,本发明的修饰mRNA的长度适于在细胞(例如,人细胞)内生产蛋白质、多肽或肽。 [0106] Generally, the length of the modified mRNA of the present invention is suitable for the production of a protein, polypeptide or peptide within a cell (e.g., human cells). 例如,mRNA的长度足以能够在细胞内翻译至少二肽。 For example, the length is sufficient to enable translation of mRNA of at least two peptides within the cell. 一个具体实施方式中,修饰mRNA的长度大于30个核苷酸。 A particular embodiment, the modified mRNA is greater than a length of 30 nucleotides. 另一个具体实施方式中,长度大于35个核苷酸。 In another specific embodiment, greater than 35 nucleotides in length. 另一个具体实施方式中,长度至少为40个核苷酸。 Another specific embodiment, the length of at least 40 nucleotides. 另一个具体实施方式中,长度至少是45个核苷酸。 In another specific embodiment, at least 45 nucleotides in length. 另一个具体实施方式中,长度至少是55个核苷酸。 In another specific embodiment, at least 55 nucleotides in length. 另一个具体实施方式中,长度至少是60个核苷酸。 In another specific embodiment, at least 60 nucleotides in length. 另一个具体实施方式中,长度至少是60个核苷酸。 In another specific embodiment, at least 60 nucleotides in length. 另一个具体实施方式中,长度至少是80个核苷酸。 In another specific embodiment, at least 80 nucleotides in length. 另一个具体实施方式中,长度至少是90个核苷酸。 In another specific embodiment, at least 90 nucleotides in length. 另一个具体实施方式中,长度至少是100个核苷酸。 In another specific embodiment, at least 100 nucleotides in length. 另一个具体实施方式中,长度至少是120个核苷酸。 In another specific embodiment, at least 120 nucleotides in length. 另一个具体实施方式中,长度至少是140个核苷酸。 In another specific embodiment, at least 140 nucleotides in length. 另一个具体实施方式中,长度至少是160个核苷酸。 In another specific embodiment, at least 160 nucleotides in length. 另一个具体实施方式中,长度至少是180个核苷酸。 In another specific embodiment, at least 180 nucleotides in length. 另一个具体实施方式中,长度至少是200个核苷酸。 In another specific embodiment, at least 200 nucleotides in length. 另一个具体实施方式中,长度至少是250个核苷酸。 In another specific embodiment, at least 250 nucleotides in length. 另一个具体实施方式中,长度至少是300个核苷酸。 In another specific embodiment, at least 300 nucleotides in length. 另一个具体实施方式中,长度至少是350个核苷酸。 In another specific embodiment, at least 350 nucleotides in length. 另一个具体实施方式中,长度至少是400个核苷酸。 In another specific embodiment, at least 400 nucleotides in length. 另一个具体实施方式中,长度至少是450个核苷酸。 In another specific embodiment, at least 450 nucleotides in length. 另一个具体实施方式中,长度至少是500个核苷酸。 In another specific embodiment, at least 500 nucleotides in length. 另一个具体实施方式中,长度至少是600个核苷酸。 In another specific embodiment, at least 600 nucleotides in length. 另一个具体实施方式中,长度至少是700个核苷酸。 In another specific embodiment, at least 700 nucleotides in length. 另一个具体实施方式中,长度至少是800个核苷酸。 In another specific embodiment, at least 800 nucleotides in length. 另一个具体实施方式中,长度至少是900个核苷酸。 In another specific embodiment, at least 900 nucleotides in length. 另一个具体实施方式中,长度至少是1000个核苷酸。 In another specific embodiment, at least 1000 nucleotides in length. 另一个具体实施方式中,长度至少是1100个核苷酸。 In another specific embodiment, at least 1100 nucleotides in length. 另一个具体实施方式中,长度至少是1200个核苷酸。 In another specific embodiment, at least 1200 nucleotides in length. 另一个具体实施方式中,长度至少是1300个核苷酸。 In another specific embodiment, at least 1300 nucleotides in length. 另一个具体实施方式中,长度至少是1400个核苷酸。 In another specific embodiment, at least 1,400 nucleotides in length. 另一个具体实施方式中,长度至少是1500个核苷酸。 In another specific embodiment, at least 1500 nucleotides in length. 另一个具体实施方式中,长度至少是1600个核苷酸。 In another specific embodiment, at least 1600 nucleotides in length. 另一个具体实施方式中,长度至少是1800个核苷酸。 In another specific embodiment, at least 1800 nucleotides in length. 另一个具体实施方式中,长度至少是20000个核苷酸。 In another specific embodiment, at least 20,000 nucleotides in length. 另一个具体实施方式中,长度至少是2500个核苷酸。 In another specific embodiment, at least 2500 nucleotides in length. 另一个具体实施方式中,长度至少是3000个核苷酸。 In another specific embodiment, at least 3,000 nucleotides in length. 另一个具体实施方式中,长度至少是4000个核苷酸。 In another specific embodiment, at least 4000 nucleotides in length. 另一个具体实施方式中,长度至少是5000个核苷酸,或大于5000个核苷酸。 In another specific embodiment, at least 5000 nucleotides in length, or more than 5000 nucleotides.

[0107] 修饰核酸的应用 [0107] Application of the modified nucleic acid

[0108] 通过本文所述方法生产的蛋白质、多肽或肽可以作为治疗性药剂用以治疗或预防一种或多种本文所述的疾病或病症。 [0108] produced by the methods described herein a protein, polypeptide, or peptide can be used as therapeutic agents for treating or preventing a disease or disorder according to one or more herein.

[0109] 治疗剂。 [0109] therapeutic agent. 提供组合物、方法、试剂盒和试剂,以治疗或预防人类和其他动物(例如,哺乳动物)的疾病或病症。 It provides compositions, methods, kits and reagents for the treatment or prophylaxis of humans and other animals (e.g., mammals) of a disease or disorder. 本发明的活性治疗剂包括由修饰核酸翻译的多肽、含有修饰核酸或由修饰核酸翻译的多肽的细胞,以及与含有修饰核酸或自修饰核酸翻译的多肽的细胞接触的细胞。 Active therapeutic agents according to the present invention comprises a nucleic acid translation modifications of the polypeptide, the cell containing a modified nucleic acid or polypeptide by modification of the translation of nucleic acids, and modified nucleic acid or modified cells from contacting a nucleic acid translated polypeptide containing cells.

[0110] 提供使用本文所述修饰核酸诱导重组多肽在细胞群内翻译的方法。 [0110] The modified nucleic acid provided herein using recombinant polypeptide translation in a cell population induced. 所述翻译可以是在体内、离体、培养基中(in culture)或在体外。 The translation may be in vivo, ex vivo, culture medium (in culture) or in vitro. 所述细胞群与含有核酸的有效量的组合物接触,所述核酸具有至少一个核苷修饰和编码重组多肽的可翻译区。 Contacting the cell population with an effective amount of a composition comprising a nucleic acid, the nucleic acid having at least one modified nucleoside untranslated region encoding the recombinant polypeptide. 所述细胞群在该核酸定位在细胞群的一个或多个细胞内且在细胞内由所述核酸翻译重组多肽的条件下发生接触。 The population of cells in which the nucleic acid is positioned in a cell or population of cells and a plurality of conditions in the intracellular polypeptide translated from the recombinant nucleic acid into contact.

[0111] 基于,至少部分地基于靶组织、靶细胞类型、给药方式、由修饰核酸翻译的蛋白质的物理性质(例如,大小)和其他决定因素,提供有效量的所述组合物。 [0111] Based, at least in part, on the target tissue, target cell type, mode of administration, the physical properties of the modified proteins translated nucleic acid (e.g., size), and other determinants, providing an effective amount of the composition.

[0112] 配制包含修饰核酸的组合物,以肌肉内给药、动脉内给药、腹膜内给药、静脉内给药、鼻内给药、皮下给药、内窥镜(endoscopically)给药、透皮给药或鞘内给药。 [0112] formulated composition comprising the modified nucleic acid to intramuscular administration, intraarterial administration, intraperitoneal administration, intravenous administration, intranasal administration, subcutaneous administration, the endoscope (endoscopically) administration, transdermal administration or intrathecal administration. 在一些具体实施方式中,组合物经配制缓释给药。 In some embodiments, a sustained release formulated composition administered.

[0113] 施用治疗剂的受试者患有疾病、病症或病况或有此风险。 Subject [0113] administration of a therapeutic agent having a disease, disorder or condition or risk. 提供有基于包括临床诊断、生物标志物水平、基因组开放式相关研究(genome-wide association study, GffAS)以及本领域已知的其他方法在内的基础而鉴别、诊断受试者并对其进行分型的方法。 There is provided on the basis of clinical diagnosis include, biomarker levels, open studies (genome-wide association study, GffAS) genomes and other methods known in the art, including the identification, diagnosis and subjected to sub-subject type approach.

[0114] 在某些具体实施方式中,所施用的由本文所述的修饰核酸翻译的重组多肽提供了在该重组多肽所施用的细胞中基本没有的功能性活性。 [0114] In certain embodiments, administration of the polypeptide translated from the recombinant nucleic acid modifications described herein provides a functional activity of the recombinant polypeptide in a cell is not substantially applied. 例如,缺少的功能性活性性质上可以是酶促的、结构性的或基因调节的活性。 For example, the lack of functional activity may be enzymatic properties, structural gene regulation or activity. [0115] 在其他具体实施方式中,所施用的重组多肽代替在重组多肽所施用的细胞中基本没有的多肽(或多个多肽)。 [0115] In other embodiments, the recombinant polypeptide in a cell is administered in place of the recombinant polypeptide in the polypeptide administered substantially free of (or polypeptides). 这种没有可以是由于编码基因或其调节通路的基因突变。 This may not be due to gene mutations encoding gene or its regulatory pathways. 或者,重组多肽的功能是拮抗存在于细胞内、细胞表面上或者自细胞分泌的内源蛋白的活性。 Alternatively, the recombinant polypeptide is to antagonize the function present in the cell, on the cell surface or secreted from the cell activity of endogenous protein. 通常,内源蛋白的活性对于受试者而言是有害的。 Typically, the activity of the endogenous protein is detrimental for the purposes of the subject. 例如,由于内源蛋白的突变而导致改变活性或定位。 For example, due to a mutation in the endogenous protein leading to alter the activity or localization. 此外,重组多肽直接地或间接地拮抗存在于细胞内、细胞表面上或者自细胞分泌的生物分子的活性。 Moreover, recombinant polypeptide directly or indirectly antagonizing present in the cell, on the cell surface or activity of biomolecules secreted from the cell. 拮抗的生物分子的示例包括脂类(例如,胆固醇)、脂蛋白(例如,低密度脂蛋白)、核酸、糖类或小分子毒素。 Exemplary biomolecules include, antagonistic lipids (e.g., cholesterol), lipoprotein (e.g., LDL), nucleic acid, carbohydrate or small molecule toxins.

[0116] 本文所述重组蛋白经设计而定位在细胞内,潜在定位在特定的细胞区室(例如细胞核)或者经设计而由细胞分泌或转移到细胞质膜上。 [0116] The recombinant protein described herein was designed to be positioned within the cell, the potential is positioned in a specific cell compartment (e.g., nuclear) designed or secreted from the cells or transferred to the plasma membrane.

[0117] 如本文所述,本发明的修饰核酸的有用特性是能够降低细胞对于外源核酸的天然免疫反应,例如,以提高蛋白产量。 [0117] As described herein, modified nucleic acid useful feature of the present invention is capable of reducing cellular innate immune response to the exogenous nucleic acid, e.g., to increase the protein production. 提供用以滴定、减少或消除细胞或细胞群内免疫反应的方法。 The method provides for the titration, reduce or eliminate the cells or cell populations of the immune response. 在一些具体实施方式中,所述细胞与包含第一剂量的第一外源核酸的第一组合物接触,并测定细胞对第一外源核酸的天然免疫反应的水平,所述第一外源核酸包含可翻译区和至少一个核苷修饰。 In some embodiments, the cells contain a first exogenous nucleic acid and a first dose of a first composition is contacted, and determining the level of the natural cellular immune response to a first exogenous nucleic acid, said exogenous first untranslated region may contain nucleic acid and at least one nucleoside modification. 随后,所述细胞与包含第二剂量的第一外源核酸的第二组合物接触,所述第二剂量具有含量比第一剂量少的第一外源核酸。 Subsequently, the cell with a first exogenous nucleic acid comprising a second dose of the second composition contacts, the second dose is less than the first dose of the content of a first exogenous nucleic acid. 或者,所述细胞与第一剂量的第二外源核酸接触。 Alternatively, contacting the cell with a second dose of the first exogenous nucleic acid. 所述第二外源核酸可以包含一个或多个可与第一外源核酸相同或不同的修饰核苷,或者,第二外源核酸可以不含修饰核苷。 Said second exogenous nucleic acid may comprise one or more identical or different from the first exogenous nucleic acid modified nucleoside, or a second exogenous nucleic acid can contain modified nucleosides. 细胞与第一组合物和/或第二组合物接触的步骤可以重复一次或多次。 Cells and the step of the first composition and / or the second composition of the contact may be repeated one or more times. 此外,可任选地测定细胞内蛋白质的生产(例如,蛋白质翻译)效率,细胞可以重复转染第一和/或第二组合物直到实现目标蛋白生产效率。 Further, the measurement may optionally be produced intracellular proteins (e.g., protein translation) efficiency, cell transfection may be repeated first and / or second composition until a protein productivity.

[0118] 疾病和病症的疗法。 [01] treatment of diseases and disorders. 提供有通过取代缺失的蛋白活性或者克服异常的蛋白活性而治疗或预防征在于缺失的或异常的蛋白质活性的疾病症状的方法。 Protein activity provided by substitution or deletion of activity of the protein against abnormal characterized in that the method of treatment or prevention of the symptoms of a disease or deleted activity of an abnormal protein.

[0119] 以功能异常或异常蛋白质活性为特征的疾病包括,但不限于癌症和增生疾病、遗传病(例如,囊性纤维化)、自体免疫疾病、糖尿病、神经退化病、心血管疾病和代谢疾病。 [0119] In dysfunction or diseases characterized by abnormal protein activity include, but are not limited to cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, and metabolic disease. 本发明提供通过引入蛋白质或基于细胞的药剂以治疗受试者的所述病症或疾病的方法,所述蛋白质或基于细胞的药剂是通过利用本文提供的修饰核酸的方法产生的,其中修饰核酸编码拮抗或者克服受试者细胞中存在的`异常蛋白质活性的蛋白质。 The present invention provides a cell-based agent into proteins or to treat a disorder or disease of the subject method, the protein or cell-based agent is a modified nucleic acid by using the methods provided herein is generated, wherein the modified nucleic acid encoding or antagonistic activity of the protein against the protein `abnormal cells present in the subject. 功能异常的蛋白质的特例是囊性纤维化跨膜转运调节物(CFTR)基因的错义突变变体,其产生导致囊性纤维化的CFTR蛋白的功能紊乱蛋白变体。 Specific examples dysfunctional protein is cystic fibrosis transmembrane conductance regulator product (CFTR) gene missense mutant variants which produce a variant protein resulting in dysfunction of the cystic fibrosis CFTR protein.

[0120] 大量疾病以缺失蛋白活性(或基本上消除蛋白活性,从而不产生正确的蛋白功能)为特征。 [0120] In a number of diseases deletion protein activity (or substantially eliminate the activity of the protein, so as not to produce the correct protein function) is characterized. 所述蛋白质可以不存在,或基本上是无功能的。 The protein may be absent, or substantially non-functional. 本发明提供了治疗受试者的上述病症或疾病的方法,所述方法通过引入或包含本文提供的修饰核酸的核酸或基于细胞的药剂,其中所述修饰核酸编码取代受试者靶细胞缺失的蛋白活性的蛋白质。 The present invention provides the above method of treating a disorder or disease in a subject, the nucleic acid or modified nucleic acid-based agent of the cell or by introducing the methods provided herein comprise, wherein the modified nucleic acid encoding a deletion target cell unsubstituted subject protein protein activity. 功能异常的蛋白的特例是囊性纤维化跨膜转运调节物(CFTR)基因的无义突变变体,其产生导致囊性纤维化的CFTR蛋白的无功能蛋白变体。 Dysfunctional proteins is a special case of the cystic fibrosis transmembrane regulator transporter (CFTR) gene nonsense mutation variant, which leads to cystic fibrosis generation of non-functional CFTR protein protein variant.

[0121] 因此,提供在细胞中存在有效量的CTFR多肽的条件下通过使受试者的细胞与修饰核酸接触而治疗哺乳动物受试者的囊性纤维化的方法,其中,所述修饰合适地包含编码功能性CFTR多肽的可翻译区。 The method under conditions [0121] Thus, presence of an effective amount of a polypeptide in a cell CTFR a subject by contacting a cell with a nucleic acid modification of treating cystic fibrosis in a mammalian subject, wherein said modified suitable comprises encoding a functional CFTR polypeptide can be translated regions. 通常的靶细胞是上皮细胞(例如肺),并且给药方式根据靶组织确定,例如,针对肺部递送,RNA分子被配制成通过吸入而给药。 The target cells are typically epithelial cells (e.g. lung), and the target tissue is determined in accordance with the mode of administration, e.g., for pulmonary delivery, RNA molecules are formulated to be administered by inhalation.

[0122] 另一个具体实施方式中,本发明提供用以治疗受试者高脂血的方法,所述方法通过向受试者的细胞群引入分拣蛋白(Sortilin,近来通过基因组研究表征的蛋白)从而改善受试者的高脂血,所述分拣蛋白是使用本文所述的方法通过编码分拣蛋白的修饰mRNA分子而产生的。 [0122] In another specific embodiment, the present invention provides a method for treating hyperlipidemia, the method by introducing protein sorting (sortilin subject to a cell population, characterized by the recent studies of histone gene ) thereby improving hyperlipidemia in a subject, the protein is sorted using the methods described herein by modifying the mRNA molecule encoding a protein produced sorting. SORTl基因编码称为分拣蛋白的反高尔基网(TGN)跨膜蛋白。 SORTl gene encodes a protein called anti-Golgi sorting network (the TGN) transmembrane protein. 遗传学研究表明,五分之一的个体具有单一核苷酸多态性,rsl2740374,在SORTl基因的1ρ13基因座,使其偏向于拥有低水平的低密度脂蛋白(LDL)和极低密度脂蛋白(VLDL)。 Genetic studies show that one in five individuals with a single nucleotide polymorphism, rsl2740374, in 1ρ13 SORTl gene locus, so tend to have low levels of low-density lipoprotein (LDL) and very low-density lipoprotein protein (VLDL). 存在于约30%的人群中的次等位基因的每一个拷贝使LDL胆固醇改变8mg/dL ;而存在于约5%的人群中的次等位基因的二个拷贝会降低LDL胆固醇16mg/dL。 Each time a copy of the allele is present in about 30% of the population of LDL cholesterol by changing 8mg / dL; and groups present in about 5% of the two copies of the allele times reduces LDL cholesterol 16mg / dL . 次等位基因的携带者还显示出40%的降低的心肌梗死风险。 Second allele carriers also shows that the risk of myocardial infarction by 40% decrease. 小鼠体内功能性研究描述了在小鼠肝脏组织中过表达SORTl导致LDL-胆固醇水平显著降低,降低80%之多,而沉默SORTl则提高LDL胆固醇接近200%(Musunuru K等,From noncoding variant to phenotype via S0RT1 at the lpl3 cholesterol locus,Nature 2010 ;466:714-721)。 Functional in vivo studies in mice is described in liver tissue of mice leads to overexpression SORTl LDL- cholesterol levels decreased significantly, as much as 80% lower, and silent SORTl LDL cholesterol is increased nearly 200% (Musunuru K, etc., From noncoding variant to via S0RT1 phenotype at the lpl3 cholesterol locus, Nature 2010; 466: 714-721).

[0123] 药物组合物 [0123] The pharmaceutical compositions

[0124] 本发明提供了由修饰mRNA产生的蛋白质,并且本文所述方法生产的蛋白质能够用于药物组合物。 [0124] The present invention provides a protein produced by a modified mRNA, and the protein produced by the methods described herein can be used in pharmaceutical compositions. 药物组合物可任选地包含一个或多个额外的治疗活性物质。 The pharmaceutical compositions may optionally contain one or more additional therapeutically active substances. 按照一些具体实施方式,提供了施用药物组合物的方法,所述药物组合物包含要递送到有需要的受试者中的一个或多个蛋白质。 DETAILED DESCRIPTION According to some embodiments, there is provided a method of administering a pharmaceutical composition, said pharmaceutical composition comprising delivering to a subject in need thereof to one or more proteins. 在一些具体实施方式中,药物组合物被施用于人。 In some embodiments, the pharmaceutical composition is administered to a human. 出于本发明的目,术语“活性成分”通常是指本文所述的蛋白质或含蛋白质的复合物。 For purposes of the present invention, the term "active ingredient" herein generally refers to a protein or protein-containing complex.

[0125] 尽管本文提供的药物组合物的描述主要指向适合施用于人的药物组合物,但本领域技术人员可以理解,所述组合物通常也适合于施用于所有种类的动物。 [0125] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for administration to humans, but those skilled in the art will appreciate, the compositions also generally suitable for administration to animals of all sorts. 改良适合于施用于人的药物组合物以使组合物适合施用于多种动物的方法是公知的,并且,兽药领域普通技术人员能仅仅通过常规的实验设计和/或实施这样的改良。 Modified suitable for administration to the human a pharmaceutical composition so that the composition is applied to a variety of methods for animals are well known, and those of ordinary skill in the art can veterinary merely by routine experimentation, design and / or implementation of such improvement. 计划给予药物组合物的受试者包括,但不限于人和/或其它灵长类;哺乳动物,包括商业相关的哺乳动物,例如牛、猪、马、绵羊、猫、狗、小鼠和/或大鼠;和/或鸟类,包括商业相关的鸟类,诸如鸡、鸭、鹅和/或火鸡。 Plan administering a pharmaceutical composition of subjects, including, but not limited to humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rat; and / or birds, including commercially relevant birds such as chickens, ducks, geese, and / or turkey.

[0126] 本文所述药物组合物的配方可以通过制药领域任何已知或今后形成的方法配制。 [0126] Formulations of the pharmaceutical compositions described herein may be formulated by any method known in the art of pharmacy or later formed. 通常,这样的配制方法包括将活性成分混入赋形剂和/或一个或多个其他附加成分的步骤,以及随后(如果有需要和/或期望的话)将所述产品成型和/或包装成预期的单剂量或多剂量单元。 In general, such methods include the formulation of the active ingredient mixed with an excipient and / or one or more additional ingredients other steps, and then (if necessary and / or desired) forming the product and / or packaging into a desired unit of single or multiple doses.

[0127] 按照本发明的药物组合物可以作为单单位剂量和/或作为多个单单位剂量成批制备、包装和/或销售。 [0127] The pharmaceutical compositions of the invention may be used as a single unit dose, and / or batch prepared as a plurality of single unit doses, packaged and / or sold. 如本文使用的,“单位剂量”是指包含预定量的活性成分的药物组合物的单份量。 As used herein, "unit dose" means a single weight comprising a predetermined amount of the active ingredient of the pharmaceutical composition. 活性成分的量通常等于施用于受试者的活性成分用量和/或此剂量的便于使用的部分用量,例如,用量的二分之一或三分之一。 The amount of active ingredient is generally equal to the amount of active ingredient administered to a subject and / or ease of use of part of the amount of this dose is, e.g., an amount of one-half or one-third.

[0128] 活性成分的相对量、药学上可接受的赋形剂和/或本发明的药物组合物中的任何额外成分将根据所治疗的受试者的特性、大小和/或条件以及更进一步根据组合物的给药途径而变化。 [0128] The relative amounts of active ingredient, pharmaceutically acceptable excipients and / or any additional ingredients in pharmaceutical compositions of the present invention according to the characteristics of the subject being treated, the size and / or conditions, and further It varies depending on the route of administration of the composition. 作为示例,组合物可以包含0.1%到100%(w/w)的活性成分。 By way of example, the composition may comprise 0.1% to 100% (w / w) of the active ingredient.

[0129] 药物组合物可以配制成额外包含药学上可接受的赋形剂,如本文所用的,所述药学上可接受的赋形剂可以包括任意和全部溶剂、分散剂、稀释剂或其他液体介质、分散或悬浮辅剂、表面活性剂、等渗剂、增稠或乳化剂、防腐剂、固体粘合剂、润滑剂等等,使其适合期望的特定剂型° Remington 的The Science and Practice of Pharmacy, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006;本文通过引证而全文引入)公开了多种用于配制药物组合物的赋形剂及已知的配制技术。 [0129] The pharmaceutical compositions may be formulated additionally comprise a pharmaceutically acceptable excipient, as used herein, the pharmaceutically acceptable excipients may include any and all solvents, dispersants, diluents, or other liquid medium, dispersing or suspending adjuvants, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, to suit the particular dosage form desired ° Remington's the Science and Practice of Pharmacy, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; and incorporated herein by reference) discloses a number of pharmaceutical compositions used in formulating excipients and techniques known in the formulation. 目前除了与物质或其衍生物不兼容的任何常规赋形剂介质(例如通过产生任何不利的生物作用或者以有害的方式与药物组合物中的其他成分相互作用)外,其用途落入本发明的范围。 Except insofar as any conventional excipients present media (e.g., interaction with other components of the pharmaceutical composition by any adverse biological effect or deleterious manner) are not compatible with the substance or a derivative thereof, their use fall within the present invention range.

[0130] 在一些具体实施方式中,药学上可接受的赋形剂的纯度至少是95%、至少是96%、至少是97%、至少是98%、至少是99%或100%。 [0130] In some embodiments, the purity of the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. 在一些具体实施方式中,赋形剂已被批准用于人类或供兽医使用。 In some embodiments, the excipient is approved for human or for veterinary use. 在一些具体实施方式中,赋形剂已经被美国食品药品管理局(UnitedStates Food and Drug Administration)批准。 In some embodiments, the excipient has been the United States Food and Drug Administration (UnitedStates Food and Drug Administration) approval. 在一些具体实施方式中,赋形剂是药品级的。 In some embodiments, the excipient is pharmaceutical grade. 在一些具体实施方式中,赋形剂符合美国药典(United States Pharmacopoeia,USP) >欧盟药典(European Pharmacopoeia, EP)、英国药典(British Pharmacopoeia)和/ 或国际药典(International Pharmacopoeia)标准。 In some embodiments, the excipient meets the standards USP (United States Pharmacopoeia, USP)> European Pharmacopoeia (European Pharmacopoeia, EP), the British Pharmacopoeia (British Pharmacopoeia) and / or the International Pharmacopoeia (International Pharmacopoeia) standards.

[0131] 用于药物组合物的制备的药学上可接受的赋形剂包括,但不限于,惰性稀释剂、分散剂和/或制粒剂、表面活性剂和/或乳化剂、崩解剂、粘合剂、防腐剂、缓冲剂、润滑剂和/或油。 [0131] Pharmaceutically acceptable for the preparation of pharmaceutical compositions excipients include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents , binders, preservatives, buffering agents, lubricants and / or oils. 这些赋形剂可选择地包含于药物组合物中。 These excipients may optionally contain in the pharmaceutical composition. 按照配方师的判断,组合物中可以有赋形剂,诸如椰子油和栓蜡、着色剂、包被剂、甜味剂、调味剂和/或赋香剂。 According to the judgment of the formulator, the compositions may have excipients such as coconut oil and suppository waxes, coloring agents, coating agents, sweetening, flavoring and / or aromatizing agent.

[0132] 示例性的稀释剂包括,但不限于,碳酸钙、碳酸钠、磷酸钙、磷酸二钙、硫酸钙、磷酸氢钙、乳糖磷酸钠、蔗糖、纤维素、微晶纤维素、高岭土、甘露醇、山梨醇、肌醇、氯化钠、干淀粉、玉米淀粉、糖粉等等,和/或它们的组合。 [0132] Exemplary diluents include, but are not limited to, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0133] 示例性的制粒剂和/或分散剂包括但不限于,土豆淀粉、玉米淀粉、木薯淀粉、羟基乙酸淀粉钠、粘土、海藻酸、瓜尔胶、柑桔渣、琼脂、斑脱土、纤维素和木制品、天然海绵、阳离子交换树脂、碳酸钙、硅酸盐、碳酸钠、交联多聚乙烯吡咯烷酮(交聚烯吡酮)、羧甲基淀粉钠(羟基乙酸淀粉钠)、羧甲基纤维素、交联羧甲基纤维素钠(交联甲羧纤维素)、甲基纤维素、预糊化淀粉(淀粉1500)、微晶淀粉、非水溶淀粉、羟甲基纤维素钙、硅酸镁铝(Veegum)、月桂基硫酸钠、季铵化合物等,和/或它们的组合。 [0133] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite earth, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinyl pyrrolidone (cross povidone), sodium carboxymethyl starch (sodium starch glycolate) , carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose cellulose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-soluble non-starch, carboxymethyl cellulose Su calcium, magnesium aluminum silicate (of veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof.

[0134] 示例性的表面活性剂和/或乳化剂包括,但不限于,天然乳化剂(例如,阿拉伯树胶、琼脂、海藻酸、海藻酸钠、黄芪胶、软骨酸、胆固醇、黄原胶、果胶、明胶、卵黄、酪蛋白、羊 [0134] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, cartilage acid, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, sheep

毛脂、胆固醇、蜡和卵磷脂)、胶质粘土(例如,斑脱土[硅酸铝]和Veegum.K'_ [硅酸镁铝])、 Wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum.K'_ [magnesium aluminum silicate]),

长链氨基酸衍生物、高分子量醇(例如,十八烷醇、十六烷醇、油醇、乙酸甘油单硬脂酸酯、乙二醇双硬脂酸酯、甘油单硬脂酸酯和丙二醇单硬脂酸酯、聚乙烯醇)、卡波姆(例如,聚亚甲基羧酸、聚丙烯酸、丙烯酸聚合物、羧基乙烯基聚合物)、角叉胶、纤维素衍生物(例如,羧甲基纤维素钠、粉末状纤维素、羟甲基纤维素、羟丙基纤维素、羟丙基甲基纤维素、甲基纤维素)、山梨糖醇酐脂肪酸酯(例如,聚氧乙烯山梨糖醇酐单月桂酸酯[Tween® 20]、聚氧乙 Long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glycerol monostearate acetate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., polymethylene acid, polyacrylic acid, acrylic acid polymer, carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., carboxymethylcellulose sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters anhydride (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene

烯山梨糖醇[Tweeif 60]、聚氧乙烯山梨糖醇酐单油酸[Tween®80]、山梨糖醇酐单棕榈酸酯[Span® 40]、山梨糖醇酐单硬脂酸酯[Span® 60]、山梨糖醇酐三硬脂酸酯[Span® 65]、甘油单油酸酯、山梨糖醇酐单油酸酯[Spanx 80]、聚氧乙烯酯(例如,聚氧乙烯单硬脂酸酯[Myq® 45]、聚氧乙烯氢化蓖麻油、聚乙氧基蓖麻油、聚氧乙烯硬脂酸酯和Solutof)、月旨肪酸糖酯、聚乙二醇脂肪酸酯(例如,CremophoP )、聚氧乙烯醚(例如,聚氧乙烯月桂醚[Brif 30])、聚乙烯吡咯烷酮、二乙二醇单月桂酸酯、三乙醇胺油酸酯、油酸钠、油酸钾、油 Alkenyl sorbitol [Tweeif 60], polyoxyethylene sorbitan monooleate, sorbitan [Tween®80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [of Span ® 60], sorbitan tristearate [Span® 65], glyceryl monooleate, sorbitan monooleate [Spanx 80], polyoxyethylene esters (e.g. polyoxyethylene monostearate aliphatic ester [Myq® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxyethylene stearate and Solutof), fatty acid sugar esters aims month, polyethylene glycol fatty acid esters (e.g. , CremophoP), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brif 30]), polyvinyl pyrrolidone, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, oleyl

酸乙酯、油酸、月桂酸乙酯、硫酸月桂酸纳、Pluronic' F68> Poloxarnefiv 188、西曲溴铵、氯化 Ethyl oleate, ethyl laurate, sodium sulfate, Pluronic 'F68> Poloxarnefiv 188, cetrimonium bromide, chloride,

十六烷基吡啶、苯扎氯铵、琥珀辛酯钠等,和/或它们的组合。 Cetylpyridinium, benzalkonium chloride, sodium docusate and the like, and / or combinations thereof.

[0135] 示例性的粘合剂包括,但不限于,淀粉(例如,玉米淀粉和淀粉糊)、凝胶、糖(例如,蔗糖、葡萄糖、右旋葡萄糖、糊精、糖蜜、乳糖、乳糖醇、甘露醇)、天然和合成的树胶(例如,阿拉伯树胶、海藻酸钠、爱尔兰藓提取物、潘瓦尔胶(panwar gum)、加特胶(ghattigum) ,isapol husk胶、羧甲基纤维素、甲基纤维素、乙基纤维素、羟乙基纤维素、羟丙基纤维 [0135] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol , mannitol), natural and synthetic gums (e.g., gum arabic, sodium alginate, extract of Irish moss, gum Pan Waer (panwar gum), gum Stuttgart (ghattigum), isapol husk gum, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose

素、羟丙基甲基纤维素、微晶纤维素、醋酸纤维素、聚乙烯吡咯烷酮、硅酸镁铝( Vbegunf) Su, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, polyvinyl pyrrolidone, magnesium aluminum silicate (Vbegunf)

和落叶松阿拉伯半乳聚糖)、海藻酸酯、聚环氧乙烷、聚乙二醇、无机钙盐、硅酸、聚甲基丙烯酸酯、蜡、水、醇等等,以及它们的组合。 And larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohols and the like, and combinations thereof .

[0136] 示例性的防腐剂可以包括,但不限于,抗氧化剂、螯合剂、抗菌性防腐剂、抗真菌性防腐剂、醇类防腐剂、酸类防腐剂和/或其他防腐剂。 [0136] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acids, preservatives and / or other preservatives. 示例性的抗氧化剂包括,但不限于,生育酚、抗坏血酸、VC棕榈酸酯(acorbyl palmitate)、丁基羟基茴香醚、丁基羟基甲苯、硫代甘油、焦亚硫酸钾、丙酸、没食子酸丙酯、抗坏血酸钠、亚硫酸氢钠、焦亚硫酸钠和/或亚硫酸钠。 Exemplary antioxidants include, but are not limited to, tocopherols, ascorbic acid, VC palmitate (acorbyl palmitate), butyl hydroxy anisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, gallic acid propylparaben, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. 示例性的螯合剂包括乙二胺四乙酸(EDTA)、一水合柠檬酸、乙二胺四乙酸二钠、乙二胺四乙酸二钾、依地酸、延胡酸、苹果酸、磷酸、依地酸钠、酒石酸和/或依地酸三钠。 Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, by sodium edetate, tartaric acid, and / or trisodium edetate. 示例性的抗菌性防腐剂包括,但不限于,苯扎氯铵、苄索氯铵、苯甲醇、溴硝醇、西曲溴銨、氯化十六烷基吡啶、氯己定、氯丁醇、氯化甲酚、氯二甲酚、甲酚、乙醇、甘油、合克替啶、咪唑烷基脲、苯酚、苯氧乙醇、苯基乙醇、硝酸苯汞、丙二醇和/或.硫柳汞。 Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, cetyl benzethonium chloride, benzyl alcohol, bronopol, cetrimide, pyridinium chloride, chlorhexidine, chlorobutanol , chlorinated cresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and / or. thiomersal. 示例性的抗真菌性防腐剂包括,但不限于,对羟基苯甲酸丁酯、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、苯甲酸、水杨酸、苯甲酸钾、山梨酸钾、苯甲酸钠、丙酸钠和/或山梨酸。 Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethylparaben, propylparaben, benzoic acid, salicylic acid, benzene potassium formate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid. 示例性的醇类防腐剂包括,但不限于,乙醇、聚乙二醇、苯酚、酚类化合物、双酚、氯代丁醇、羟基苯甲酸酯和/或苯乙醇。 Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. 示例性的酸类防腐剂包括,但不限于,维生素A、维生素C、维生素Ε、β-胡萝卜素、柠檬酸、乙酸、脱氢乙酸、抗坏血酸、山梨酸和/或植酸。 Exemplary acids preservatives include, but are not limited to, vitamin A, vitamin C, vitamin Ε, β- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid and / or phytic acid. 其他防腐剂包括,但不限于,生育酚、生育酚乙酯、deteroxime mesylate、西曲溴铵、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)、乙二胺、十二烷基硫酸钠(SLS)、月桂基乙醚硫酸钠(SLES)、亚硫酸氢钠、焦亚 Other preservatives include, but are not limited to, tocopherol, tocopherol, ethyl, deteroxime mesylate, cetrimide, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, dodecyl sodium alkyl sulfate (the SLS), sodium lauryl ether sulfate (the SLES), sodium bisulfite, metabisulfite

硫酸钠、亚硫酸钾、焦亚硫酸钾、Glydant plus'®、Phenonip®、对轻基苯甲酸甲酯、GeraialP115> Germall* I1、Neolone™、Kathon™ 和/ 或Euxyl®。 Sodium sulfate, potassium sulfite, potassium metabisulfite, Glydant plus'®, Phenonip®, mild methyl benzoate, GeraialP115> Germall * I1, Neolone ™, Kathon ™ and / or Euxyl®.

[0137] 示例性的缓冲剂包括,但不限于,柠檬酸缓冲液、醋酸缓冲液、磷酸缓冲液、氯化铵、碳酸钙、氯化钙、柠檬酸钙、葡乳醛酸钙、葡庚糖酸钙、葡萄糖酸钙、D-葡糖酸、甘油磷酸钙、乳酸钙、丙酸、乙酰丙酸钙、戊酸、磷酸氢二钙、磷酸、磷酸钙、磷酸氢钙、醋酸钾、氯化钾、葡萄糖酸钾、钾混合物、磷酸氢二钾、磷酸二氢钾、磷酸钾混合物、醋酸钠、碳酸氢钠、氯化钠、柠檬酸钠、乳酸钠、磷酸氢二钠、磷酸二氢钠、磷酸钠混合物、三羟甲基氨基甲烷、氢氧化镁、氢氧化招、海藻酸、无热原水、等渗盐水、林格氏溶液(Ringer' s solution)、乙醇等,和/或它们的组合。 [0137] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, gluceptate calcium gluconate, calcium gluconate, D- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, calcium phosphate, calcium phosphate, calcium hydrogen phosphate, potassium acetate, chloride potassium gluconate, potassium mixtures, dibasic potassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate , a mixture of sodium phosphate, tris, magnesium hydroxide, strokes, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution (Ringer 's solution), ethanol and the like, and / or their combination. [0138] 示例性的润滑剂包括,但不限于,硬脂酸镁、硬脂酸钙、硬脂酸、二氧化硅、滑石、麦芽、甘油二十二烷酸酯、氢化植物油、聚乙二醇、苯甲酸钠、乙酸钠、氯化钠、亮氨酸、十二烷基硫酸续、十烧基硫酸纳等等,以及它们的组合。 [0138] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene alcohol, sodium benzoate, sodium acetate, sodium chloride, leucine, sodium lauryl sulfate continued ten burning yl sodium sulfate and the like, and combinations thereof.

[0139] 示例性的油类包括,但不限于,扁桃、杏仁、鳄梨、巴巴苏、佛手柑、黑加仑籽、琉璃苣、杜松、春黄菊、菜籽、香菜、巴西棕榈、蓖麻、肉桂、可可油、椰子、鱼肝、咖啡、玉米、棉籽、鸸鹋、桉树、月见草、鱼、亚麻籽、香叶醇、葫芦、葡萄籽、榛子、牛膝草、豆蘧酸异丙酯、荷荷巴、奇异果、醒目薰衣草(Iavandin)、薰衣草(lavender)、朽1檬、山鸡椒、澳洲坚果、锦葵、芒果籽、白绒花籽、水紹、肉豆蘧、橄榄、柑桔、罗非鱼(orange roughy)、棕榈、棕榈仁、桃仁、花生、I!粟籽、南瓜子、油菜籽、米糠、迷迭香、红花、檀香、sasquana、香薄荷、沙棘、芝麻、黄油、娃酮、大豆、向日葵、茶树、蓟、椿、香根草、胡桃和麦胚芽油。 [0139] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black currant seed, borage, cade, camomile, canola, caraway, carnauba, castor , cinnamon, cocoa butter, coconut, fish liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl Qu beans propyl, jojoba, kiwi, eye-catching lavender (Iavandin), lavender (lavender), rotten 1 lemon, pheasant pepper, macadamia nut, mallow, mango seed, Cashmere seeds, water Shao, Qu meat, beans, olives , citrus, tilapia (orange roughy), palm, palm kernel, peach kernel, peanut, I! poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn , sesame seeds, butter, baby ketone, soybean, sunflower, tea tree, thistle, piles, vetiver, walnut, and wheat germ oil. 不例性的油类包括,但不限于,硬脂酸丁酯、辛酸甘油三酯、癸酸甘油三酯、环甲基硅油、癸二酸二乙酯、二甲基硅油360,豆蘧酸异丙酯、矿物油、辛基十二烷醇、油醇、硅酮油和/或它们的组合。 Examples of oils do not include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, beans Qu acid isopropyl, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0140] 用于口服或肠胃外给药的液体剂型包括,但不限于,药学上可接受的乳液、微乳液、溶液、悬液、糖浆和/或酏剂。 Liquid dosage forms [0140] for oral or parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and / or elixirs. 除活性成分以外,液体剂型可以包括通常用于本领域的惰性稀释剂,例如,水或其他溶剂,增溶剂和乳化剂,例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、二甲基甲酰胺、油类(特别地,棉籽、落花生、玉米、胚芽、橄榄、蓖麻和芝麻油)、甘油、四氢糠醇、聚乙二醇和山梨聚糖脂肪酸酯和它们的组合。 Addition to the active ingredient, the liquid dosage forms may comprise inert diluents commonly used in the art, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and combinations thereof. 除了惰性稀释剂,口服组合物还可以包括佐剂,例如润湿剂、乳化剂和悬浮剂、甜味剂、调味剂和/或香香剂。 Besides inert diluents, the oral compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and / or savory agents. 在胃肠外给药的某些具体实施方式中,组合物可以与增溶剂,例如 In certain embodiments for parenteral administration, the compositions may solubilizers, e.g.

Cremopho1.' 醇类、油类、改性油类、二元醇、聚山梨醇酯、环式糊精、多聚物和/或它们的 Cremopho1. 'Glycols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or their

组合混合。 Mixed combination.

[0141] 注射剂,例如无菌注射水性或油性悬浮液,可以依据已知技术利用合适的分散剂、润湿剂和或悬浮剂配制。 [0141] Injections, for example, sterile injectable aqueous or oily suspensions, using suitable dispersing agents may be according to known techniques, or wetting agents and suspending agents. 无菌注射剂可以是无毒的胃肠道外可接受的稀释剂和/或溶剂中的无菌注射溶液、悬浮液和/或乳剂,例如,1,3- 丁二醇溶液。 The sterile injectable preparation may be a non-toxic parenterally acceptable diluent and / or solvent sterile injectable solutions, suspensions and / or emulsions, e.g., 1,3-butylene glycol. 可使用的可接受媒介和/溶剂是水、林格氏溶液、USP和等渗氯化钠溶液。 Acceptable medium and / solvents that may be employed are water, Ringer's solution, USP and isotonic sodium chloride solution. 通常可使用无菌的非挥发性油作为溶剂或悬浮介质。 Typically sterile, non-volatile oil as a solvent or suspending medium. 出于这个目的,可以使用任何温和的非挥发性油,包括合成的甘油单酯或甘油二酯。 For this purpose, any bland fixed oils including synthetic mono- or diglycerides. 脂肪酸(例如油酸)可用于注射制剂。 Fatty acids (e.g., oleic acid) can be used in the preparation of injectables.

[0142] 注射组合物可以例如通过细菌阻留性滤器过滤和/或通过引入灭菌剂而除菌,所述灭菌剂是无菌固体组合物的形式,并且可以在使用前溶解于或分散于无菌水或其他无菌注射介质中。 [0142] Injectable compositions can, for example, by filtration through a bacterial-retaining filter properties and / or sterilized by the sterilizing agent introduction, the sterilizing agent is in the form of sterile solid compositions and may be dissolved or dispersed prior to use in sterile water or other sterile injectable medium.

[0143] 为了延长活性成分的作用,常常希望延缓皮下注射或肌肉内注射的活性成分的吸收。 [0143] In order to prolong the effect of the active ingredient, it is often desirable to slow the absorption of subcutaneous or intramuscular injection of the active ingredient. 这可以通过使用水溶性差的结晶或无定形材料的液体悬液而实现。 This can be achieved by the use of poorly water-soluble liquid suspension of crystalline or amorphous material. 药物的吸收速率取决于其溶解速率,而溶解速率可依赖于其晶体大小和结晶形式。 The rate of absorption of the drug depends upon its rate of dissolution, and the dissolution rate thereof may depend upon crystal size and crystalline form. 或者,可以通过将药物溶解或悬浮于油性媒介中而实现胃肠道外施用药物的延缓稀释。 Alternatively, by dissolving or suspending the drug in an oil medium to achieve dilution retard drug administered parenterally. 通过在生物可降解多聚物(例如聚乳酸聚乙醇酸交酯)中形成药物的微囊基质,可以制备药效持久的可注射组合物。 By forming microencapsule matrices of the drug in biodegradable polymers (e.g., polylactic polyglycolide), the lasting effect of the injectable compositions can be prepared. 根据药物和多聚物的比例和所使用的特定多聚物的性质,可以控制药物释放速率。 Depending on the nature and proportion of the drug and the particular polymer used in the polymer, the rate of drug release can be controlled. 其他生物可降解多聚物的实例包括多聚原酸酯和聚酸酐。 Other biodegradable polymers include poly-orthoesters and polyanhydrides. 可以通过将药物包裹入和身体组织兼容的脂质体或是微乳液而配制或制备药效持久的可注射组合物。 By entrapping the drug into the body tissue compatible, and in liposomes or microemulsions formulated lasting effect or injectable composition.

[0144] 用于直肠或阴道给药的组合物一般是栓剂,其可以通过将组合物与适宜的非刺激性赋形剂(例如可可油、聚乙二醇或栓蜡)混合配置而成,所述赋形剂在室温下是固体,但在体温下是液态,从而在直肠或阴道径中是融化状态并释放活性成分。 [0144] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by the composition with a suitable non-irritating excipient (e.g., cocoa butter, polyethylene glycol or suppository wax) configured by mixing, the excipients are solid at room temperature but liquid at body temperature, so that a molten state and release the active ingredient in the rectum or vaginal diameter.

[0145] 用于口服给药的固态剂型包括胶囊、片剂、丸剂、粉剂和粒剂。 [0145] The solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. 在所述固态剂型中,活性物质与至少一个惰性的药学上可接受的赋形剂(例如柠檬酸钠或磷酸氢二钙)和/或填料或增量剂(例如,淀粉、乳糖、鹿糖、葡萄糖、甘露醇和硅酸),粘合剂(例如,羟甲基纤维素、海藻酸盐、凝胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶),保湿剂(例如甘油),崩解剂(例如,琼脂、碳酸钙、土豆或木薯淀粉、海藻酸、某些硅酸盐和碳酸钠),溶解延迟剂(例如,石蜡),吸收促进剂(例如,四铵化合物),润湿剂(例如,十六醇和甘油单硬脂酸酯),吸附剂(例如,高岭土、班脱土)和润滑剂(例如,滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠)和它们的组合混合。 The solid dosage forms, the active substance and at least one pharmaceutically acceptable inert excipients (such as sodium citrate or dicalcium phosphate) and / or fillers or extenders (e.g., starch, lactose, sugar deer , glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. , agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., tetra ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), adsorbents (e.g., kaolin, bentonite) and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sodium sulfate) and mixed combinations thereof. 在胶囊、片剂和丸剂的情况下,剂型可以包含缓冲剂。 In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0146] 可以采用相似类型的固体组合物作为软和硬填料凝胶胶囊中的填料,所述胶囊使用了诸如乳糖或奶糖以及高分子量聚乙二醇等的赋形剂。 [0146] a similar type may be employed as solid compositions of fillers in soft and hard gelatin capsule filling, the capsule using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. 可以用包衣或包壳(例如肠衣或制药领域已知的其他包衣)配制固体剂型的片剂、糖衣丸、胶囊、丸剂和粒剂。 The solid dosage forms may be formulated with a coating or cladding (e.g., enteric or other coatings known in the pharmaceutical art) tablets, dragees, capsules, pills, and granules. 所述固体剂型可任选包含乳浊剂,并且可以是任选以延迟方式仅向或优选向肠道的特定部分释放活性成分的组合物。 The solid dosage form may optionally comprise opacifying agents, and can optionally be released in a delayed manner to a particular part of the intestinal tract, the active ingredient only or preferably to the composition. 可以使用的包埋组合物的示例包括多聚物和蜡。 Examples of embedding compositions which can be used include polymers and waxes. 可以采用相似类型的固体组合物作为软和硬填料凝胶胶囊中的填料,所述胶囊使用了诸如乳糖或奶糖以及高分子量聚乙二醇等的赋形剂。 Similar type may be employed as solid compositions of fillers in soft and hard gelatin capsule filling, the capsule using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0147] 用于外用和/或透皮施用组合物的剂型可以包括膏剂、糊剂、霜剂、洗剂、凝胶、粉齐U、溶液、喷雾、吸入剂和/或贴剂。 [0147] for the topical and / or transdermal dosage composition may be administered include ointments, pastes, creams, lotions, gels, powders Qi U, solutions, sprays, inhalants and / or patches. 通常地,当需要时,活性成分可以在无菌条件下与药学上可接受的赋形剂和/或任何需要的防腐剂和/或缓冲剂混合。 Generally, when necessary, with the pharmaceutically active ingredient may be in a pharmaceutically acceptable excipient, under sterile conditions, and / or any preservatives and / or buffers as needed. 此外,本发明还涉及透皮贴剂的使用,所述透皮贴剂通常具有向身体提供化合物的缓释的额外优势。 The present invention further relates to the use of transdermal patches, the transdermal patch generally has the additional advantage of providing a sustained release of the compound to the body. 此类剂型可以通过,例如将化合物溶解和/或分散在正确的介质中而制备。 Such dosage forms can be, for example, prepared by dissolving the compound and in the correct medium / or dispersing. 可选地或者另外,可以通过提供速度控制膜和/或将化合物分散到多聚物基质和/或凝胶中对速度进行控制。 Alternatively or additionally, the control may be dispersed in the film and / or the speed of the compound by providing a polymer matrix and / or gel for speed control.

`[0148] 适合在递送本文所述的皮内药物组合物中使用的装置包括短针装置,例如在US4, 886,499、US5, 190,521、US5, 328,483、US5, 527,288、US4, 270,537、US5, 015,235、US5, 141,496和US5,417,662专利中描述的那些。 `[0148] apparatus suitable for use in the transdermal delivery of the pharmaceutical compositions described herein include short needle devices was, for example, in US4, 886,499, US5, 190,521, US5, 328,483, US5, 527,288 , US4, 270,537, US5, 015,235, US5, 141,496, and those described in patent US5,417,662. 可以通过限制针有效刺入皮肤的长度的装置,例如在国际公开号为W099/34850中描述的装置或及其功能等效物,施用皮下组合物。 By limiting the effective penetration length of the skin needle device, for example, in International Publication No. devices or their functional equivalents in W099 / 34850 is described, the composition is administered subcutaneously. 合适的装置有将液体组合物经液体喷射注射器和/或经刺入角质层并产生到达真皮的喷射流的针递送至真皮的喷射注射装置。 Suitable means has the liquid composition is a liquid jet injector and / or via a needle piercing the stratum corneum to the dermis and to generate the jet delivered to the dermis jet injection device. 喷射注射装置描述在,例如US5,480,381、US5, 599,302、US5, 334,144、US5, 993,412、US5, 649,912、US5, 569,189、US5, 704,911、US5, 383,851、US5, 893,397、US5, 466,220、US5, 339,163、US5, 312,335、US5, 503,627、US5, 064,413、US5, 520,639、US4, 596,556、US4, 790,824、US4, 941,880、US4, 940,460 美国专利和国际公开号为W097/37705和W097/13535中。 Jet injection devices described for example in US5,480,381, US5, 599,302, US5, 334,144, US5, 993,412, US5, 649,912, US5, 569,189, US5, 704,911, US5 , 383,851, US5, 893,397, US5, 466,220, US5, 339,163, US5, 312,335, US5, 503,627, US5, 064,413, US5, 520,639, US4, 596 , 556, US4, 790,824, US4, 941,880, US4, 940,460 US and international patent publication number W097 / 37705 and W097 / 13535 in. 合适的装置有使用压缩气体以促进粉末形式的疫苗穿过皮肤表层到达真皮的冲击性粉末/颗粒递送装置。 Suitable powder impact apparatus used with a pressurized gas to facilitate vaccine in powder form through the skin to the dermis surface / particle delivery devices. 可选择地或者另外,可以将常规注射器用在皮内给药的经典曼托方法中。 Alternatively or additionally, conventional syringes may be used in the classical methods within the Mantoux skin of administration.

[0149] 配制用于外用给药的组合物包括,但不限于,液体和/或半液体制剂,例如擦剂、洗剂、水包油和/或油包水乳剂,例如霜剂、膏剂和/或糊剂,和/或溶液和/或悬液。 The composition [0149] formulated for topical administration include, but are not limited to, liquid and / or semi-liquid preparations such as liniments, lotions, oil in water and / or water in oil emulsions such as creams, ointments and / or pastes, and / or solutions and / or suspensions. 例如,可以配制包含大约1%到大约10%(w/w)活性成分的外用组合物,尽管活性成分的浓度可能高至活性成分在该溶剂中的溶解度界限。 For example, the formulation comprises about 1% to about 10% (w / w) of the composition for external use of the active ingredient, although the concentration of active ingredient may be as high as the solubility limit of active ingredient in the solvent. 配制的外用组合物可以进一步包含本文所述的一个或多个附加的活性成分。 Formulated topical composition described herein may further comprise one or more additional active ingredients.

[0150] 可以配制、制备、包装和/或销售通过口腔进行肺部给药的药物组合物。 [0150] can be formulated, prepared, packaged and / or sold for pulmonary administration via the oral pharmaceutical composition. 可以将这样的组合物配制成包含含有活性成分并且直径在约0.5nm到约7nm之间或在约Inm到约6nm之间的干颗粒。 Such compositions may be formulated to contain the active ingredient and comprising a diameter between about 0.5nm to about 7nm or dry particles between about Inm to about 6nm. 所述组合物便利地为干粉形式,用于利用包括干粉储器的装置(推进剂的气流可以指向所述干粉储器从而分散干粉)给药或利用自推进溶剂/粉末分散容器(例如包含溶解和/或悬浮在密封容器中的低沸点推进剂中的活性成分的装置)给药。 The composition is conveniently in the form of a dry powder, means for utilizing comprises a dry powder reservoir (propellant gas flow may be directed to disperse the powder reservoir dry powder) administration or use of self-propelling solvent / powder dispensing container (e.g., comprising dissolving means and / or suspended in a sealed container of a low boiling point propellant active ingredient) is administered. 所述粉剂包括颗粒,其中以重量计至少98%的颗粒的直径大于0.5nm且以数量计至少95%的颗粒的直径小于7nm。 Such powders comprise particles wherein at least 98% by weight of a particle diameter larger than 0.5nm diameter and at least 95% by number of the particles are less than 7nm. 或者,以重量计至少95%的颗粒的直径大于Inm且以数量计至少90%的颗粒的直径小于6nm。 Alternatively, at least 95% by weight of the particles of a diameter greater than the diameter of Inm and at least 90% by number of the particles are less than 6nm. 干粉组合物可以包括固体细粉稀释剂(例如糖)且便利地以单位剂量形式提供。 Dry powder compositions may include a solid fine powder diluent (e.g. sugar) and conveniently be presented in unit dosage form.

[0151] 低沸点推进剂通常包括在大气压下沸点低于65 °?的液体推进剂。 [0151] Low boiling propellants generally include a boiling point below 65 °? Propellant liquid at atmospheric pressure. 通常,推进剂可以占组合物的大约50%到大约99.9%(w/w),活性成分可可占组合物的大约0.1%到大约20%(w/w)。 Typically, the propellant may comprise about 50% to about 99.9% (w / w) of the composition, the active ingredients comprise from about 0.1% cocoa to about 20% of the composition (w / w). 推进剂可以进一步包括额外的成分,例如液态非离子和/或固态阴离子表面活性剂和/或固态稀释剂(其颗粒大小可以与包含活性成分的颗粒具有同等级)。 Propellant may further comprise additional ingredients, such as liquid non-ionic and / or solid anionic surfactant and / or a solid diluent (which may have the same level of particle size and the particle comprising the active ingredient).

[0152] 配制的用于肺部给药的药物组合物可以提供溶液和/或悬液的微滴形式的活性成分。 [0152] The pharmaceutical compositions formulated for pulmonary delivery may provide a solution and the active ingredient in the form of droplets / or suspension. 所述组合物可以作为包含活性成分、任选无菌的水溶液和/或稀释醇溶液和/或悬液形式配制、制备、包装和/或出售,并且可以利用任何喷雾和/或雾化装置方便地给药。 The composition may comprise as an active ingredient, optionally a sterile aqueous and / or dilute alcoholic solutions and / or suspensions in the form of formulating, manufacturing, packaging, and / or sold, and may utilize any nebulization and / or atomization device convenient administered. 所述组合物还可以进一步包含一个或多个额外的成分所述成分包括,但不限于,芳香剂、例如糖精钠、挥发油、缓冲剂、表面活性剂和/或防腐剂例如甲羟基苯甲酸盐(酯)。 The composition may further comprise one or more additional ingredients including the ingredients, but not limited to, flavoring agents, for example sodium saccharin, volatile oils, buffering agents, surfactants and / or preservatives such as methyl hydroxybenzoate salt (ester). 通过这种给药途径提供的微滴的平均直径可以在大约0.1nm到大约200nm之间。 The average diameter of the droplets provided by this route of administration may be between about 0.1nm to about 200nm.

[0153] 本文所述用于肺部给药的配方也用于药物组合物的鼻内给药。 [0153] The formulations for pulmonary administration are also described herein for intranasal administration of a pharmaceutical composition. 另一个配制用于鼻内给药的组合物是包含活性成分且平均颗粒为0.2 μ m到500 μ m的粗粉。 Another composition is formulated for intranasal administration comprising the active ingredient and having an average particle of 0.2 μ m to 500 μ m coarse powder. 所述组合物经配制用于以吸鼻烟的方式(即,通过鼻道从靠近鼻子手持的粉末容器快速吸入)给药。 The composition is formulated in a manner snuff (i.e., from a hand-held powder container close to the nose rapid inhalation through the nasal passage) administration.

[0154] 配制用于鼻腔给药方式的组合物可以,例如包含大约少至0.l%(w/w)到多至100%(w/w)的活性成分,并且可以包含本文所述的一个或多个其他成分。 The composition [0154] formulated for nasal administration may, for example, comprise from about as little as 0.l% (w / w) to as much as 100% (w / w) of the active ingredient, and may comprise the herein one or more other ingredients. 可以配制、制备、包装和/或销售药物组合物,用于口腔给药。 It can be formulated, prepared, packaged and / or sold in a pharmaceutical composition, for oral administration. 例如,所述组合物可以使用常规方法配制成片剂和/或锭剂的形式,且可以含有例如0.1%到20%(w/w)的活性成分,包含口服可溶的和/或可降解组合物的主要部分(balance)和任选的本文所述的一个或多个额外成分。 For example, the composition may be formulated using conventional methods into the form of tablets and / or lozenges and may contain from 0.1% to 20% (w / w) active ingredient e.g., oral administration comprising soluble and / or degradable one or more additional ingredients described (Balance), and optionally a major portion of the composition herein. 或者,配制的用于口腔给药的组合物可以包括含有活性成分的粉末化和/或烟雾化和/或喷雾化的溶液和/或悬液。 Alternatively, the composition is formulated for oral administration may comprise a powder and / or smoke and / or spraying of a solution containing the active ingredient and / or suspensions. 在被分散时,所述粉末化、烟雾化和/或喷雾化的组合物的平均颗粒和/或微滴大小可以在大约0.1nm到大约200nm的范围内,并且可以进一步包含一个或多个本文所述的额外成分。 When dispersed, the powdered, smoke and / or spray of mean particle composition and / or the droplet size may range from about 0.1nm to about 200nm, and may further comprise one or more article said additional ingredients.

[0155] 可以配制、制备、包装和/或销售药物组合物,用于眼部给药。 [0155] can be formulated, prepared, packaged and / or sold in a pharmaceutical composition, for ophthalmic administration. 例如,所述组合物可以配制成滴眼液的形式,所述滴眼液含有例如0.1/lv.0%(w/w)活性成分的水性或油性液体赋形剂的溶液和/或悬液。 For example, the composition can be formulated in the form of ophthalmic solution, the ophthalmic solution containing, for example an aqueous or oily liquid 0.1 / lv.0% (w / w) of the active ingredient the excipient solutions and / or suspensions . 所述滴液可以进一步包含缓冲剂、盐和/或本文所述的一个或多个其他任意额外成分。 The drops may further comprise one or more additional ingredients any of the other buffers, salts and / or described herein. 其他可使用的眼部给药组合物包括含有微晶形式的活性成分和/或在脂质体制剂中的活性成分的组合物。 Other ocular administration the composition may include a composition comprising the active ingredient in microcrystalline form and / or the active ingredient in a liposomal agent. 滴耳液和/或滴眼液被认为是落入本发明的范围。 Ear drops and / or eye drops are considered to fall within the scope of the invention. [0156] 药物制剂的配制和/或制备的常规思路可见于,例如Remington:The Science andPractice of Pharmacy 21st 出版社、Lippincott Williams & Wilkins, 2005 年(本文通过参考而全文引入)中。 [0156] formulated in a pharmaceutical formulation and / or preparation of conventional thinking may be found in, for example, Remington: The Science andPractice of Pharmacy 21st Press, Lippincott Williams & Wilkins, 2005 (incorporated herein by reference) in.

[0157] 给药 [0157] administration

[0158] 本发明提供包括向有需要的受试者施用通过本文所述方法生产的蛋白或组合物的方法。 [0158] The present invention provides a process for the production of protein by the methods described herein or composition administered to a subject in need thereof. 可以使用可有效预防、治疗、诊断疾病或病况和/或病症(例如,与工作记忆缺失(working memory deficits)有关的疾病、病况和/或病症)或使其成像的任意数量和任意给药方法向受试者施用蛋白或复合物,或其药物、成像、诊断或预防组合物。 It can be effective in preventing, treating, diagnosing a disease or condition and / or disorder (e.g., working memory deficits (working memory deficits) related diseases, conditions and / or disorders) or made of any method of administration and any number of imaging administration of proteins or complexes thereof, or a pharmaceutical, imaging, diagnosis or prevention composition to a subject. 所需准确用量根据受试者的物种、年龄和整体情况、疾病的严重程度、特定组合物及其给药方式、活性方式等等而随受试者变化。 The precise amount required depending on the species, age, and the overall situation, the severity of the disease, the particular composition and its mode of administration, the active mode and the like with the subject changes. 一般将按照本发明的组合物配制成剂量单位形式以便于给药和统一用量。 Generally formulated in dosage unit form composition according to the present invention is to facilitate administration and uniformity of dosage. 尽管如此,可以理解的是,本发明组合物的日用总量可以由主治医在可靠的医学判断范围内决定。 Nevertheless, it is understood that the daily total composition of the present invention may be determined by the attending health within the scope of sound medical judgment. 特定病人的特定治疗效果、预防效果或合适的成像剂量水平随多种因素变化,所述因素包括待治疗的病症和病症的严重程度;使用的特定化合物的活性;使用的特定组合物;病人的年龄、体重、整体健康状况、性别和饮食情况;给药的时间;给药的方式,和使用的特定化合物的排泄率;治疗的时间长度;与使用的特定化合物联合或同时使用的药物;以及医学领域已知的类似因素。 Particular therapeutic effect to a particular patient, a suitable imaging or prophylactic effect dose level changes depending on various factors, the factors including the severity of the disorder and the condition to be treated; activity of the specific compound employed; the specific composition used; patient the age, body weight, general health, sex, and diet; time of administration; the mode of administration, and rate of excretion of the particular compound used; the length of time of the treatment; combined with the particular compound or pharmaceutical use; and like factors known in the medical field.

[0159] 递送的蛋白质和/或其药物、预防、诊断或成像组合物可以施用于动物,例如哺乳动物(例如,人类、驯化动物、猫、狗、小鼠、大鼠及其他)。 [0159] The delivery of protein and / or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered to animals such as mammals (e.g., humans, domesticated animals, cats, dogs, mice, rats and others). 在一些具体实施方式中,其药物、预防、诊断或成像组合物施用于人。 In some embodiments, pharmaceutical, prophylactic, diagnostic, or imaging composition to a person.

[0160] 根据本发明的待递送蛋白质和/或其药物、预防、诊断或成像组合物可以通过任何方式给药。 [0160] The delivery of the present invention to be a protein and / or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered by any means. 在一些具体实施方式中,蛋白质和/或其药物、预防、诊断或成像组合物可以通过一种或多种途径给药,所述途径包括口服、静脉内、肌内、动脉内、髓内、鞘内、皮下、心室内、透皮、皮内、直肠、阴道内、腹膜内、外用(例如,通过粉剂、油膏、霜齐U、凝胶、洗液和/或滴剂)、粘膜、鼻、口腔、肠、玻璃体、瘤内、舌下;通过气管滴注法、支气管滴注法和/或吸入法;作为空腔喷雾,鼻喷雾,和/或气溶胶,和/或通过门静脉导管。 In some embodiments, the protein and / or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered by one or more of routes including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams Qi U, gels, lotions and / or drops), mucosal, nose, mouth, intestine, vitreous, intratumoral, sublingual; by tracheal instillation, bronchial instillation, and / or inhalation; cavity as a spray, nasal spray and / or aerosol, and / or through a portal vein catheter . 在一些具体实施方式中,蛋白质或复合物,和/或其药物、预防、诊断或成像组合物可以通过系全身静脉注射给药。 In some embodiments, a protein or complex, and / or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be systemically administered by intravenous injection system. 特定具体实施方式中,蛋白质或复合物,和/或其药物、预防、诊断或成像化合物可以静脉内和/或口服给药。 Particular embodiment, the protein or complex, and / or pharmaceutical, prophylactic, diagnostic, or imaging / or compounds may be administered orally and intravenously. 特定具体实施方式中,蛋白质或复合物,和/或其药物、预防、诊断或成像组合物可以使蛋白质或复合物穿过血脑屏障、血管屏障或其他上皮屏障的方式给药。 Particular embodiment, the administration of proteins or complexes, and / or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be a protein or complexes cross the blood barrier or other vascular epithelial barrier manner.

[0161] 不论如何,本发明包含通过任何考虑到在药物递送科学可能的进步的合适方式递送蛋白质或复合物,和/或其药物、预防、诊断或成像化合物。 [0161] In any case, the present invention comprises a delivery by any suitable manner taking into account the possible scientific advances in drug delivery or protein complexes, and / or pharmaceutical, prophylactic, diagnostic, or imaging compound.

[0162] 通常,给药的最适方式依赖于多种因素,包括蛋白质或含有与至少一个待递送的物质相结合的蛋白质的复合物的特性(例如在胃肠道、血液等环境中的稳定性),病人的状态(例如,病人是否能忍受特定的给药方式)。 [0162] Generally, the most suitable mode of administration depends on a variety of factors, including proteins or characteristics (e.g., stable in the gastrointestinal tract, blood or the like with the environment containing at least one substance to be delivered in conjunction with a protein complex sexual), condition of the patient (e.g., whether the patient is able to tolerate a particular mode of administration). 本发明包括通过考虑到药物递送科学可能的进步的合适方式递送所述药物、预防、诊断或成像组合物。 Delivered by the present invention includes a drug delivery suitable manner taking into account the possible scientific progress of the drug, prophylactic, diagnostic, or imaging composition.

[0163] 某些具体实施方式中,按照本发明的组合物可以以每天足以递送以受试者体重计大约0.0001mg/kg 到大约100mg/kg,0.01mg/kg 到大约50mg/kg,大约0.lmg/kg 到大约40mg/kg,大约0.5mg/kg 到大约30mg/kg,大约0.0 lmg/kg 到大约10mg/kg,大约0.lmg/kg到大约10mg/kg或大约lmg/kg到大约25mg/kg,每天一次或多次,以达到期待的治疗、诊断、预防或成像效果的剂量水平给药。 [0163] In certain specific embodiments, the compositions according to the invention may be sufficient to deliver the subject body weight per day to about 0.0001mg / kg to about 100mg / kg, 0.01mg / kg to about 50mg / kg, about 0 .lmg / kg to about 40mg / kg, about 0.5mg / kg to about 30mg / kg, from about 0.0 lmg / kg to about 10mg / kg, from about 0.lmg / kg to about 10mg / kg or from about lmg / kg to about 25mg / kg, once or more daily, to achieve the expected therapeutic, diagnostic, prophylactic or imaging effect dose level administered. 可以每天三次、每天两次、每天一次、每隔一天、每隔两天、每周、每两周、每三周或每四周递送期待的剂量。 May be three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks or every four weeks to deliver anticipated dose. 某些具体实施方式中,利用多次给药而递送期待的剂量(例如,二、三、四、五、六、七、八、九、十、十一、十二、十三、十四或更多次给药)。 In certain embodiments, the use of multiple doses expected to be delivered dose (eg, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations).

[0164] 蛋白质或复合物可以与一个或多个其他治疗、预防、诊断或成像药剂联合使用。 [0164] or protein complexes can be one or more other therapeutic, prophylactic, diagnostic imaging agents, or in combination. 所述“联合”,并非意味着药剂必须同时给药和/或配制成用于一起递送,尽管这些递送方法也落在本发明的范围内。 The "joint" does not mean that the drug must be administered and / or formulated for delivery together, although these methods of delivery are also within the scope of the invention fall. 组合物的给药可以与一个或多个其他期待的治疗或医学过程同时、在先或随后发生。 The composition may be administered with one or more other therapeutic or medical procedures expected simultaneous, preceding or subsequent. 通常,按照药物的确定剂量和/或时间表施用每种药剂。 Typically, according to the determined dose and / or schedule of administration of each agent. 在一些具体实施方式中,本发明包括与改善其生物利用度、减少和/或调节其代谢、抑制其排出、和/或调解其体内分布的药剂联合递送所述药物、预防、诊断或成像组合物。 In some embodiments, the present invention includes to improve its bioavailability, reduce and / or modulate their metabolism, inhibit their discharge, and / or agents which mediate delivery and distribution in the combined medicament, prophylactic, diagnostic, or imaging composition thereof. 特定的具体实施方式中,提供包含一个或多个修饰核酸与诱导抗体依赖性细胞毒性的蛋白质的联合治疗,其中,所述修饰核酸包含可翻译区,所述可翻译区编码促进哺乳动物受试者免疫性的蛋白质。 Particular embodiments, there is provided a nucleic acid comprising one or antibody dependent cellular cytotoxicity and induce more modified proteins of combination therapy, wherein said nucleic acid comprises modified untranslated region, the coding region may facilitate translation mammalian subject by immunity protein. 例如,提供包括一个或多个编码曲妥珠单抗的核酸和粒细胞集落刺激因子(G-CSF)的治疗。 For example, there is provided comprising one or more encoding nucleic acids trastuzumab and granulocyte colony stimulating factor (G-CSF) treatment. 这种联合治疗对于针对曲妥珠单抗产生诱导抗性的Her2+胸腺癌患者特别有用(参见,例如,Albrecht, Immunotherapy.2 (6): 795-8 (2010))。 This combination is particularly useful for the treatment of Her2 + breast cancer patients have induced resistance against trastuzumab (see, for example, Albrecht, Immunotherapy.2 (6): 795-8 (2010)).

[0165] 可以进一步理解,联合使用的治疗、预防、诊断或成像活性药物可以以单个组合给药或以不同组合分别给药。 [0165] may be further appreciated that the use of combined treatment, prevention, diagnosis or imaging of the active drug may be administered separately or in a single composition is administered in different combinations. 通常,预期用于联合给药的药剂以不超过其单独使用时的剂量水平的水平使用。 Typically, the agent contemplated for combined administration in a dose level not exceeding the level of use thereof alone. 在一些具体实施方式中,用于联合给药的水平低于其单独给药时的剂量水平。 Level In some embodiments, a combined administration dosage level which is lower than administered alone.

[0166]自联合方案中采用的治疗(疗法或过程)的特定组合需要考虑所需治疗和/或过程的兼容性和要达到的预期治疗效果。 [0166] Since the particular combination regimen employed in combination therapies (therapeutics or procedures) need to consider the compatibility and the desired treatment and the desired therapeutic effect to be achieved and / or processes. 还应理解,所采用的治疗对于相同病症可以达到预期的效果(例如,按照本发明的用于治疗癌症的组合物可以与化疗药物同时给药),或者其可达到不同的效果(例如,任何不良效果的控制)。 It should also be appreciated that the therapy employed may achieve the desired effect for the same disorder (e.g., according to the present invention, compositions for treating cancer may be administered simultaneously with the chemotherapeutic agent), or they may achieve different effects (e.g., any poor control effect).

[0167] 试剂盒 [0167] Kit

[0168] 本发明提供多种用于方便地和/或有效地实现本发明方法的试剂盒。 [0168] The present invention provides for more easily and / or efficiently implemented method of the present invention is a kit. 例如,本文所述的是用于利用本文所述修饰核酸生产蛋白质的试剂盒。 For example, described herein are for use herein the modified nucleic acid kit for protein production. 一般,试剂盒包含足够量和/或大量组分以允许使用者完成受试者的重复治疗和/或完成多次实验。 Typically, the kit contains a sufficient amount and / or a large number of components to allow the user to complete the repeated treatment of the subject and / or completion of several experiments.

[0169] 定义 [0169] defined

[0170] 治疗药物(therapeutic agent):术语“治疗药物”是指当施用给受试者时,具有治疗、诊断和/或预防效果和/或得到预期的生物学和/或药学效果的任何药物。 [0170] Drug treatment (therapeutic agent): The term "therapeutic agent" means that when administered to a subject, has a therapeutic, diagnostic and any pharmaceutical / or prophylactic effect and / or give the desired biological and / or pharmaceutical effect .

[0171] 动物:如同本文所用,术语“动物”是指动物中的任何一种。 [0171] Animal: As used herein, the term "animal" means any animal. 在某些具体实施方式中,“动物”是指处于任何发育阶段的人类。 In some embodiments, "animal" refers to humans in any stage of development. 在某些具体实施方式中,“动物”是指处于任何发育阶段的非人动物。 In certain embodiments, "animal" refers to non-human animals at any stage of development. 某些的具体实施方式中,非人动物是哺乳动物(例如,啮齿动物、小鼠、大鼠、兔、猴、狗、猫、绵羊、牛、灵长类或猪)。 Certain specific embodiments, the non-human animal is a mammal (eg, rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, pigs or primates). 在一些具体实施方式中,动物包括,但不限于,哺乳动物、鸟类、爬虫类、两栖类、鱼类和虫类。 In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and insects. 在一些具体实施方式中,动物是转基因动物,基因工程动物或克隆动物。 In some embodiments, the animal is a transgenic animal, genetically engineered animals or animal cloning.

[0172] 近似:如同本文所用,术语“近似”或“大约”,当用于一个或多个目标值时,是指近似于所述参照值的值。 [0172] Approximate: As used herein, the term "approximately" or "about", when used in the one or more target value refers to a value approximate to the reference value. 某些具体实施方式中,除非明确指出或根据上下文显而易见(除了所述数超过100%的可能值),术语“近似”或“大约”是指在任一方向上(大于或少于)落入所示参考值的25%、20%、29%、18%、17%、16%、15%、14%、13%、12%、11%、10%、9%、8%、7%、6%、5%、 In certain specific embodiments, unless explicitly stated or apparent from the context (the number of possible values ​​of more than 100% in addition), the term "approximately" According or "approximately" means either direction (greater than or less than) falls shown 25% of the reference value, 20%, 29%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% , 5%,

4%、3%、2%、1%或更少的数值范围。 4%, 3%, 2%, 1% or less of the range of values.

[0173] 结合:如同本文所用,当用于两个或更多物质时,术语“结合”、“共轭”、“相连”、“螯合”和“连接”表示物质直接或者通过作为连接物的一个或多个其他物质彼此物理结合或连接,以形成足够稳定的结构,从而使物质在使用该结构的条件(例如生理条件)下保持物理 [0173] in combination: As used herein, when two or more substances used, the term "binding", "conjugate", "connected", "chelating" and "connected" indicates that the substance directly or through a linker one or more other substances bound or physically connected to each other, enough to form a stable structure, so that the substance held in the use condition of the physical structure (e.g., physiological conditions)

彡口口◦ San mouth ◦

[0174] 生物学活性:如同本文所用,短语“生物学活性”是指任意物质在生物系统和/或生物体中有活性的性质。 [0174] Biological activity: As used herein, the phrase "biological activity" refers to any of the nature of the active substances in a biological system and / or organism. 例如,当向生物体给药时,对所述生物体有生物学作用的物质被认为是有生物学活性的。 For example, when administered to an organism, the organism has a biological effect of a substance is considered to be biologically active. 特定具体实施方式中,当核酸是具有生物学活性的时,具有整条核酸的至少一个生物学活性的核酸部分一般被称作“生物学活性”部分。 In particular embodiment, when the nucleic acid is biologically active, at least one biologically active portion of a nucleic acid having the entire nucleic acid is generally referred to as "biological activity" section.

[0175] 保守:如同本文所用,术语“保守的”是指多核苷酸序列或氨基酸序列各自的核苷酸或氨基酸残基,所述残基在所比较的两条或更多相关序列的相同位点不发生改变。 [0175] Conservative: As used herein, the term "conservative" refers to a polynucleotide or amino acid sequences of the respective nucleotide or amino acid residue, the same or more residues in the two sequences being compared in site does not change. 相对保守的核苷酸或氨基酸是在较序列其他位置出现的核苷酸或氨基酸更相关的序列中保守的核苷酸或氨基酸。 Relatively conserved nucleotides or amino acids are conserved nucleotide or amino acid sequence of nucleotides or amino acids than in the other positions appear more relevant sequence. 在一些具体实施方式中,如果两条或更多序列彼此具有100%同一性,则被称作“完全保守”。 In some embodiments, if two or more sequences with 100% identity to each other, were called "fully conserved." 在一些具体实施方式中,如果两条或更多序列彼此具有至少70%同一性、至少80%同一性、至少90%同一性或至少95%同一性,则被称作“高度保守”。 In some embodiments, if two or more sequences having at least 70% identity to each other, at least 80% identity, at least 90% identity or at least 95% identity, were referred to as "highly conserved." 在一些具体实施方式中,如果两条或更多序列彼此具有大约70%同一性、大约80%同一性、大约90%同一性、大约95%同一性、大约98%同一性或大约99%同一性,则被称为“高度保守”。 In some embodiments, if two or more sequence identity to each other by about 70%, about 80% identity, about 90% identity, about 95% identity, about 98% or about 99% identity to the same sex, is called "highly conservative." 在一些具体实施方式中,如果两条或更多序列彼此之间具有至少30%同一性、至少40%同一性、至少50%同一性、至少60%同一性、至少70%同一性、至少80%同一性、至少90%同一性或至少95%同一性则被称为“保守”。 In some embodiments, if two or more sequences having at least 30% identity to each other, at least 40% identity, at least 50% identity, at least 60% identity, at least 70% identity, at least 80 % identity, at least 90% identity or at least 95% identity are known as "conservative." 在一些具体实施方式中,如果两条或更多序列彼此之间具有大约30%同一性、大约40%同一性、大约50%同一性、大约60%同一性、大约70%同一性、大约80%同一性、大约90%同一性、大约95%同一性、大约98%同一性或大约99%同一性则被称作“保守”。 In some embodiments, if two or more sequences having about 30% identity to each other, about 40% identity, about 50% identity, about 60% identity, about 70% identity, about 80 % identity, about 90% identity, about 95% identity, about 98% identity, or about 99% identity were called "conservative."

`[0176] 表达:如同本文所用,核酸序列的“表达”是指一个或多个以下事件:(I)从DNA序列产生RNA模板(例如,通过转录);(2)加工RNA转录本(例如,剪接、编辑、5'加帽和/或3'末端处理);(3)将RNA翻译成多肽或蛋白;以及(4)多肽或蛋白的翻译后修饰。 `[0176] Expression: As used herein," expression "nucleic acid sequence refers to one or more of the following events: (the I) generating an RNA template (e.g., by transcription) from the DNA sequence; (2) processing of an RNA transcript (e.g. , splicing, editing, 5 'capping and / or 3' end processing); and post-translation modifications (4) polypeptide or protein; and (3) the translation of the RNA into a polypeptide or protein.

[0177] 离体:如同本文所用,“离体”是指事件在生物体外发生,例如,在生物体外的人工环境中的组织内或组织上,例如,对天然条件具有最小改变。 [0177] In vitro: As used herein, "isolated" refers to an event occurring in vitro, e.g., in vitro artificial environment in the tissue or tissues, e.g., with minimal changes to the natural conditions.

[0178] 有功能的:如同本文所用,“有功能的”生物分子是以显示以之表征的性质和/或活性的形式的生物分子。 [0178] with a function: As used herein, "functional" biological molecule is displayed to the nature and form of representation / or activity of a biological molecule.

[0179] 同源:如同本文所用,术语“同源”是指聚合分子之间(例如,核酸分子(例如,DNA分子和/或RNA分子)之间和/或多肽分子之间)的总的关联整体相关性。 [0179] Homologous: As used herein, the term "homologous" refers to a (e.g., between a nucleic acid molecule between the polypeptide molecules (e.g., DNA molecule, and / or RNA molecules) and / or a) between the total polymeric molecules associated with the overall relevance. 在一些具体实施方式中,如果聚合分子的序列有至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%或至少99%的同一性,则认为其彼此“同源”。 In some embodiments, if the sequence of the molecule by polymerization of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, which is considered as being "homologous." 在一些具体实施方式中,如果聚合分子的序列有至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少99%相似,则认为其彼此“同源”。 In some embodiments, if the sequence of the molecule by polymerization of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% similarity, which is considered as being "homologous." 术语“同源”必然涉及至少两条序列(核苷酸序列或氨基酸序列)之间的比较。 The term "homologous" necessarily involves a comparison between (nucleotide or amino acid sequence) of at least two sequences. 按照本发明,如果其编码的多肽中至少约20个氨基酸的至少一段有至少约50%同一性、至少约60%同一性、至少约70%同一性、至少约80%同一性或至少约90%同一性,则认为两条核苷酸同源。 According to the present invention, if it encodes a polypeptide at least about 20 amino acids in at least one section of at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 80% identity, or at least about 90 % identity, two nucleotide homology is considered. 在一些具体实施方式中,通过编码至少4-5个独特的氨基酸的片段的能力鉴定同源核苷酸序列。 In some embodiments, homologous nucleotide sequences identified by a unique capacity of at least 4-5 amino acids of the encoded fragments. 对于待考虑同一性的核苷酸序列,必须考虑这些氨基酸彼此相对的同一'I"生和临近空间(approximate spacing)两者。对于长度少于60个核苷酸的核苷酸序列,通过编码至少4-5个独特氨基酸的片段的能力确定同源性。按照本发明,如果蛋白质的至少约20个氨基酸至少一段之间具有至少约50%同一性、至少约60%同一性、至少约70%同一性、至少约80%同一性或至少约90%同一性,则认为这两条蛋白序列是同源的。 Identity to the nucleotide sequence to be considered, these amino acids must be considered the same both opposing 'I "green and near space (approximate spacing) with each other for less than 60 nucleotides in length of a nucleotide sequence, by encoding ability unique fragment of at least 4-5 amino acid homology is determined. according to the present invention, if at least one piece between at least about 20 amino acids of a protein having at least about 50% identity, at least about 60% identity, at least about 70 % identity, at least about 80% identity, or at least about 90% identity, it is considered that two sequences are homologous proteins.

[0180] 同一性:如同本文所用,术语“同一性”是指聚合分子(例如,核酸分子(例如,DNA分子和/或RNA分子)之间和/或多肽分子之间)的整体相关性。 [0180] Identity: As used herein, the term "identity" refers to a polymeric molecule (e.g., and / or between polypeptide molecules or between nucleic acid molecules (eg, DNA molecules and / or RNA molecules)) in the overall correlation. 例如,计算两条核酸序列的同一性百分数可以通过,例如出于最佳比较目的而对齐所述两条序列(例如,出于最佳对齐目的在第一和第二核酸序列之一或两条中都引入间隔,为比较的目的而可以忽视不相同的序列)而进行。 For example, percent identity of two nucleic acid sequences can be calculated by, for example, for optimal comparison purposes aligning the two sequences (e.g., for purposes of optimal alignment of one of the first and second nucleic acid sequences or two We are incorporated in the interval for the comparison purposes the same sequence not negligible) is performed. 某些具体实施方式中,出于比较目的对齐的序列的长度是参照序列长度的至少30%、至少40%、至少50%、至少60%、至少70%、至少80%、至少90%、至少95%或100%。 Certain specific embodiments, the length of the sequence aligned for comparison purposes is the length of the reference sequence is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. 随后比较相应核苷酸位置的核苷酸。 Then compares the corresponding nucleotide position. 当第一序列中的位置被与第二序列相应位点的核苷酸相同的核苷酸所占据时,则该位置的分子是相同的。 When a position in the first sequence and a second nucleotide sequence corresponding sites occupied by identical nucleotides, then the molecules are identical at that position. 两条序列之间的同一性百分数是考虑了出于两条序列最佳对齐的目的而需要引入的间隔的数量和每个间隔的长度后,序列间共有的相同位点的数量的函数。 The percent identity between the two sequences is a consideration of the number and length of the interval for each interval of the two sequences are optimally aligned for purposes need to be introduced, a function of the number of shared sequence between the same sites. 两条序列间的序列比较和同一性百分数的计算可以利用数学算法实现。 Comparison of sequences and calculation of percent identity between two sequences can be achieved using a mathematical algorithm. 例如,两条序列间的同一'I"生百分数可以利用如Computational MolecularBiology, Lesk, AM编,牛津大学印制,纽约,1988 年;Biocomputing:1nformatics andGenome Projects, Smith, D.ff.编,Academic Press,纽约,1993 年;Sequence Analysisin Molecular Biology, von Heinje, G, Academic Press, 1987 年;Computer Analysis ofSequence Data,部分I, Griffin, AM和Griffin, HG编,Humana Press,新泽西,1994 年;以及Sequence Analysis Primer, Gribskov, M.和Devereux, J.编,M Stockton Press,纽约,1991年描述的方法测定,以上全部在此通过引证而全文引入。例如,两条核苷酸序列间的同一性百分数可以利用已被嵌入使用PAM120加权余数表(间隔长度罚分为12,间隔罚分为4)的ALIGN 程序(2.0 版)的Meyers 和Miller 算法(CABI OS, 1989 年,4:11-17)测定。或者,两条核苷酸序列之间的同一性百分数也可以通过利用NWS gapdna.CMP矩阵的GCG软件包中的GAP程序测定。 通常用于测 For example, the same percentage of students' I "between two sequences can be used as Computational MolecularBiology, Lesk, AM, ed., Oxford University printing, New York, 1988; Biocomputing: 1nformatics andGenome Projects, Smith, D.ff. ed, Academic Press , New York, 1993; Sequence Analysisin Molecular Biology, von Heinje, G, Academic Press, 1987 years; Computer Analysis ofSequence Data, part I, Griffin, AM, and Griffin, HG eds, Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds, M Stockton Press, New York, 1991 described a method of measurement, all of the above is hereby incorporated by reference. For example, the percent identity between two nucleotide sequences can be has been embedded using a PAM120 weight residue table (gap length penalty of 12, gap penalty of 4) in the ALIGN program (Version 2.0) algorithm of Meyers and Miller (CABI OS, 1989 year 4: 11-17) was measured. Alternatively, the percent identity between two nucleotide sequences can also be determined by using the GAP program in the GCG software package NWS gapdna.CMP matrix is ​​usually used to measure 序列间同一性百分数的方法包括,但不限于在Carillo, H.和Lipman, D.,SIAM J Applied Math., 48:1073 (1988 年)(在此通过引证而全文引入)中公开的那些。用于测定同一性的技术被编码成公众可获得的计算机程序。用于测定两条序列间同源性的示例性计算机软件包括,但不限于,GCG程序包,Devereux, J.等,Nucleic Acids Research, 12(I),387 (1984年)),BLASTP、BLASTN和FASTA Atschul, SF等,J.Molec.Biol.,215,403 (1990 年))。 The method of percent identity between sequences include, but are not limited to Carillo, H., and Lipman, D., SIAM J Applied Math, 48:. 1073 (1988 years) (hereby incorporated by reference) those disclosed. technique for determining identity is encoded into a publicly available computer programs. for determining homology between two sequences exemplary computer software including, but not limited to, GCG program package, Devereux, J., etc., Nucleic Acids Research, 12 (I), 387 (1984 years)), BLASTP, BLASTN, and FASTA Atschul, SF, etc., J.Molec.Biol., 215,403 (1990 year)).

[0181] 抑制基因表达:如同本文所用,短语“抑制基因表达”表示导致基因表达产物数量的减少。 [0181] inhibition of gene expression: As used herein, the phrase "inhibition of gene expression" means the product resulting in reduction in the number of gene expression. 表达产物可以是自基因转录的RNA(例如,mRNA)或自基因转录的mRNA所翻译的多肽。 Expression product may be RNA transcribed from a gene (eg, mRNA) or polypeptide mRNA transcribed from the translation. mRNA水平的降低一般导致由此翻译的多肽水平的降低。 Reducing the mRNA levels typically result in reduced levels of polypeptides translated therefrom. 可以通过用于测定mRNA或蛋白质的标准技术测定表达水平。 The expression level can be determined by standard techniques for measuring mRNA or protein.

[0182] 体外:如同本文所用,术语“体外”是指发生在人工环境(例如,试管或反应器、细胞培养物、皮氏培养皿等)的事件,而非在生物体内(例如,动物、植物或微生物)。 [0182] In vitro: As used herein, the term "in vitro" refers to an event occurring in an artificial environment (e.g., a test tube or reactor, cell culture, Petri dishes, etc.), rather than in vivo (e.g., animal, plants or microorganisms).

[0183] 体内:如同本文所用,术语“体内”是指发生在生物体内(例如,动物、植物或微生物)的事件。 [0183] vivo: As used herein, the term "in vivo" refers to events in vivo (e.g., animal, vegetable or microbial) a.

[0184] 分离的:如同本文所用,术语“分离的”是指物质或实体已经(I)从在产生之初与之相关的至少一些成分中分离(不论天然环境或试验环境),和/或(2)人工进行生产、制备和/或制造。 [0184] Isolated: As used herein, the term "isolated" refers to a substance or entity that has been (I) separation (whether natural environment or test environment) from at least some of the components associated with the beginning of the generation, and / or (2) artificial production, preparation and / or manufacture. 分离的物质和/或分子可以自产生之初与之相关的至少约10%、约20%、约30%、约40%、约50%、约60%、约70%、约80%、约90%或更多的其他成分中分离。 At least about 10% isolated substances and / or molecules may be generated from the beginning of the associated, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about separation of 90% or more of the other components. 在一些具体实施方式中,分离的物质有大于约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或超过99%的纯度。 In some embodiments, the isolated substance is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99% or more than 99% pure. 如同本文所用,如果基本上不含其他化合物,则物质是“纯”的。 As used herein, if substantially free of other compounds, the substance is a "pure".

[0185] 预防:如同本文所用,术语“预防”是指部分地或彻底地延缓特定疾病、病况和/或病症的发作;部分地或彻底地延缓特定疾病、病况和/或症状的一个或多个症状、特征或临床表现的发作(例如,在可鉴别的疾病、病况和/或病症之前);部分地或彻底地延缓从潜在疾病、病况和/或症状发展到活跃的疾病、病况和/或病症;和/或减少发展成为与特定疾病、病况和/或症状相关的病理的风险。 [0185] Prevention: As used herein, the term "prevention" refers to a particular disease, condition onset partially or completely delaying and / or condition; partially or completely delaying a particular disease, condition and / or symptoms or symptom, characteristics or clinical manifestations of seizures (for example, before identifiable diseases, conditions and / or disorders); partially or completely defer from underlying diseases, conditions and / or symptoms develop into active disease, conditions and / or condition; and / or reduce the risk of developing a particular disease, condition and / or symptoms associated pathology.

[0186] 相似性:如同本文所用,术语“相似性”是指多聚物分子之间的整体相关性,例如,核酸分子(例如,DNA分子和/或RNA分子)之间和/或多肽分子之间。 [0186] Similarity: As used herein, the term "similarity" refers to the overall correlation between the polymer molecules, e.g., and / or between polypeptide molecules nucleic acid molecules (eg, DNA molecules and / or RNA molecules) between. 如本领域所理解的,除了计算相似性百分数考虑保守取代外,可以通过与计算同一性百分数相同的方式计算多聚物分子彼此之间的相似性百分数。 As is understood in the art, except that the percent similarity is calculated considering conservative substitutions, the percentage similarity between each polymer molecule may be calculated by calculating percent identity in the same manner.

[0187] 受试者:如同本文所用,术语“受试者”或“病人或患者”是指例如,出于实验、诊断、预防和/或治疗目的的,可以施用按照本发明的组合物的任意的生物体。 [0187] subject: As used herein, the term "subject" or "patient or patient" means, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes, the composition may be administered in accordance with the present invention any organism. 典型的受试者包括动物(例如,哺乳动物,如小鼠、 大鼠、兔、非人灵长类和人)和/或植物。 Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans), and / or plants.

[0188] 基本上:如同本文所用,术语“基本上”是指目标特征或性质表现整个或近乎整个程度(extend或degree)的定性情况。 [0188] substantially: As used herein, the term "substantially" refers to a certain characteristic or property performance of the entire or nearly the entire extent (extend or degree) qualitative situation. 生物领域普通技术人员理解,生物和化学现象几乎不会,如果有的话,达到完成和/或进行到完全或者达到或避免绝对结果。 Biological skilled in the art understand that biological and chemical phenomena rarely, if ever, reached the completion and / or proceed to completeness or achieve or avoid an absolute result. 因此术语“基本上”本文用于表示许多生物和化学现象内在具有的潜在缺乏的完全性(completeness)。 Thus, the term "substantially" is used herein to mean a lot of inherent biological and chemical phenomena have a complete (completeness) lack of potential.

[0189] 遭受或患有:“患有”疾病、病况和/或病症的个体被诊断为患有或表现出疾病、病况和/或病症的一个或多个症状。 [0189] suffer or suffer from: "suffering from" individual diseases, conditions and / or disorders are diagnosed disease, a condition and / or one or more symptoms of illness as having or showing.

[0190] 易感或易患:对疾病、病况和/或病症“易感”的个体未被诊断患有和/或可能没有显不出疾病、病况和/或病症的症状。 [0190] susceptible or predisposed: individual diseases, conditions and / or disorders "susceptible" is not diagnosed with and / or may not be nearly as good as the disease, the symptoms of the condition and / or disorder. 在一些具体实施方式中,对于疾病、病况和/或病症(例如,癌症)易感的个体可能由以下一个或多个进行表征:(I)与疾病、病况和/或病症形成有关的基因突变;(2)与疾病、病况和/或病症有关的遗传多态性;(3)与疾病、病况和/或病症有关的蛋白质和/或核酸活性和/或表达的提高和/或降低;(4)与疾病、病况和/或病症形成有关的习惯和/或生活方式;(5)疾病、病况和/或病症的家族史;以及(6)接触和/或感染了与疾病、病况和/或病症有关的微生物。 In some embodiments, for diseases, conditions and / or disorders (e.g., cancer) susceptible individuals may be characterized by one or more of the following: (I) and diseases, conditions and / or disorders related to mutations formed ; (2) genetic polymorphisms associated with diseases, conditions and / or disorders; and or and or enhance (3) associated with the diseases, conditions and / or disorders of protein / nucleic acid activity / expression and / or decreased; ( 4) is formed with a disease, conditions and / or disorders related to diet and / or lifestyle; (5) diseases, conditions and family / or condition; and (6) contact and / or infected with diseases, conditions and / related microbes or conditions. 在一些具体实施方式中,对于疾病、病况和/或病症易感的个体会罹患疾病、病况和/或病症。 In some embodiments, for diseases, conditions and / or disorders susceptible individual will be suffering from a disease, condition and / or disorder. 在一些具体实施方式中,对于疾病、病况和/或病症易感的个体不会罹患疾病、病况和/或病症。 In some embodiments, for diseases, conditions and / or disorders susceptible to an individual not suffering from a disease, condition and / or disorder.

[0191] 治疗有效量:如同本文所用,术语“治疗有效量”表示当施用于患有或易感于疾病、病况和/或病症的受试者时,待递送药剂(例如,核酸、药物、治疗药剂、诊断药剂、预防药剂等等)的量足以治疗、改善疾病、病况和/或病症的症状,诊断、预防和/或延缓其发作。 [0191] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means that when administered to a subject suffering from or susceptible to a disease, condition and / or disorder, an agent to be delivered (e.g., nucleic acids, drugs, the amount of therapeutic agents, diagnostic agents, drug prevention, etc.) sufficient to treat, ameliorate symptoms of disease, conditions and / or disorders, diagnosis, prevention and / or delay the onset.

[0192] 转录因子:如同本文所用,术语“转录因子”是指调节(例如,通过激活或抑制转录)DNA转录成RNA的DNA结合蛋白。 [0192] transcription factor: As used herein, the term "transcription factor" refers to a condition (e.g., by activating or inhibiting transcription of) the DNA is transcribed into RNA DNA binding proteins. 某些转录因子单独影响转录调节,而其他则于其他的蛋白质共同起作用。 Some transcription factors alone affects the transcription regulation, while others act together to other proteins. 某些转录因子在一定的条件下既可以激活转录也可以抑制转录。 Some transcription factor under certain conditions may be possible to suppress the transcriptional activation of transcription. 通常,转录因子结合特定靶序列或与靶基因的调节区中的特定共有序列高度相似的序列。 Typically, transcription factor binding specific target sequence or the regulatory regions of target genes specific sequences are highly similar to the consensus sequence. 转录因子可以单独调节靶基因的转录或与其他分子构成复合物而调节。 Transcription factors regulate transcription of target genes alone or with other molecules constituting the composite was adjusted.

[0193] 治疗:如同本文所用,术语“治疗”是指部分地或彻底地缓解、改善、改进、解除特定疾病、病况和/或症病症,延缓其发作,抑制其发展,降低其严重度,和/或减少其一个或多个症状、特征、或临床表现的发生。 [0193] Treatment: As used herein, the term "treating" refers to partially or completely alleviate, ameliorate, improve, releasing the particular disease, condition and / or disease condition, delaying the onset of, inhibit the development of, reducing the severity of, and / or reducing one or more symptoms, features, or clinical presentation occurs. 例如,“治疗”癌症可以是指抑制肿瘤的存活、生长和/或扩散。 For example, "treating" may refer to inhibit tumor cancer survival, growth and / or proliferation. 治疗可以是施用于没有出现疾病、病况和/或病症的征兆(例如,在可鉴别的疾病、病况和/或病症之前)的受试者,和/或出于降低发展成与疾病、病况和/或病症相关的病理的风险的目的,施用于仅出现疾病、病况和/或病症早期征兆的受试者。 Disease, condition, symptoms and / or condition being treated may be administered to no (e.g., can be identified prior to diseases, conditions and / or disorders) subject, and / or for reducing the development of and to the disease, condition, and subject / object risk or disorders associated pathologies, appears only applied to diseases, conditions and / or disorders of the early signs. 在一些具体实施方式中,治疗包括向有需要的受试者递送与治疗活性核酸有关的蛋白质。 In some embodiments, the treatment comprises the delivery of therapeutic nucleic acids to the activity of a protein associated with a subject in need thereof.

[0194] 非修饰的:如同本文所用,“非修饰的”是指修饰前的核酸。 [0194] Non-Modified: As used herein, "non-modified" refers to a nucleic acid before modification.

实施例 Example

[0195] 实施例1:合成修饰的mRNA Synthesis of Modified mRNA: [0195] Example 1

[0196] 本发明的修饰的mRNA(modRNA)是利用标准实验室方法和材料而制备的。 [0196] modified according to the present invention, mRNA (modRNA) was prepared using standard laboratory methods and materials. 感兴趣的基因的开放阅读框(ORF)两侧为包含强Kozak翻译起始信号的5'非翻译区(UTR)和0-球蛋白3'^1?,所述α-球蛋白3'UTR以用作polyA加尾模板的oligo (dT)序列结尾。 The open reading frame of the gene of interest (ORF) flanked by 5 comprise a strong Kozak translation initiation signal 'untranslated region (UTR) and from 0 to 3 globulin' ^ 1 ?, the α- globin 3'UTR in polyA tailed oligo as a template (dT) sequence end. 用假尿苷(Ψ)和5-甲基-胞苷(5meC)修饰modRNA,以减少细胞天然免疫反应的。 With pseudouridine ([Psi]) and 5-methyl - cytidine (5meC) modified modRNA, in order to reduce the cellular innate immune response. KarikoK等,Immunity23:165-75 (2005年),Kariko K等,Mol Ther 16:1833-40 (2008年),AndersonBR 等,NAR(2010 年)。 KarikoK etc., Immunity23: 165-75 (in 2005), Kariko K, etc., Mol Ther 16: 1833-40 (in 2008), AndersonBR etc., NAR (2010 years).

[0197] 克隆、基因合成和载体测序由DNA2.0Inc (Menlo Park,CA)完成。 [0197] Cloning, Gene synthesis and sequenced by a carrier DNA2.0Inc (Menlo Park, CA). 载体序列和插入序列如SEQ ID NOs: 5-8所示。 The insert and vector sequences sequences of SEQ ID NOs: 5-8 shown in FIG. 利用XbaI或Hind III对ORF进行限制性酶切消化并且利用加尾-PCR用于cDNA合成。 Restriction digestion of the ORF and used -PCR using tailed cDNA synthesis using XbaI or Hind III. 该加尾-PCR的cDNA产物作为修饰的mRNA合成反应的模板使用,所述合成反应利用修饰的核苷混合物(修饰的U/C由TriLink Biotech, San Diego,CA 制造,未修饰的A/G 自Epicenter Biotechnologies, Madison, WI 购买)和CellScriptMegaScript™(Epicenter Biotechnologies,Madison, WI)完全mRNA 合成试剂盒,其中每种核苷的浓度为25mM。 The cDNA was tailed -PCR product as a template modification using mRNA synthesis reaction, the synthesis reaction using a mixture of modified nucleosides (modified U / C by the TriLink Biotech, San Diego, CA manufacture, unmodified A / G from Epicenter Biotechnologies, Madison, WI later) and CellScriptMegaScript ™ (Epicenter Biotechnologies, Madison, WI) complete mRNA synthesis kit, wherein the concentration of each nucleoside was 25mM. 该体外转录反应在37°C下进行3-4小时。 The in vitro transcription reaction was carried out at 37 ° C 3-4 hours. PCR反应使用HiFi PCR2XMaster Mix™(Kapa Biosystems, Woburn, MA)。 PCR reactions using HiFi PCR2XMaster Mix ™ (Kapa Biosystems, Woburn, MA). 该体外转录的mRNA产物进行琼脂糖凝胶电泳并显像。 The in vitro transcribed mRNA product was subjected to agarose gel electrophoresis and imaging. 利用Ambion/Applied Biosystems (Austin, TX)MEGAClear RNA™ 纯化试剂盒纯化mRNA。 MEGAClear ™ RNA was purified using mRNA Purification Kit Ambion / Applied Biosystems (Austin, TX). 利用PureLink™ PCR 纯化试剂盒(Invitrogen, Carlsbad, CA)或PCR cleanup 试剂盒(Qiagen, Valencia, CA)进行PCR。 Using PureLink ™ PCR Purification Kit (Invitrogen, Carlsbad, CA) or PCR cleanup kit (Qiagen, Valencia, CA) for PCR. 产物在Nanodrop™UV Absorbance (ThermoFisher,Waltham,MA)上定量。 The product in quantitative Nanodrop ™ UV Absorbance (ThermoFisher, Waltham, MA). 产物的质量、紫外吸收性质和可视化在1.2%琼脂糖凝胶上进行。 Quality of the product, and the UV-absorbing properties for visualization on a 1.2% agarose gel. 产物在TE缓冲液中重悬。 The product was resuspended in TE buffer.

[0198] 利用酵母Poly (A)聚合酶(Affymetrix, Santa Clara, CA)对带有腺苷类似物的修饰mRNA 进行poly (A)加尾。 [0198] yeast Poly (A) polymerase (Affymetrix, Santa Clara, CA) with a modified mRNA of the adenosine analogs is poly (A) tailing. 利用HiFi PCR2X Master Mix™(Kapa Biosystems, Woburn,MA)进行PCR反应。 PCR reactions were performed using the HiFi PCR2X Master Mix ™ (Kapa Biosystems, Woburn, MA). 利用重组痘病毒加帽酶(New England BioLabs, Ipswich, MA)和重组2' -氧-甲基转移酶(Epicenter Biotechnologies, Madison, WI)对修饰RNA 进行转录后加帽,以产生5' -鸟苷Capl结构。 Use of recombinant poxvirus capping enzyme (New England BioLabs, Ipswich, MA) and recombinant 2 '- O - methyl transferase enzyme (Epicenter Biotechnologies, Madison, WI) modified RNA transcription after capping, to produce 5' - Bird glycosides Capl structure. Cap2结构和Cap3结构可以利用额外的2' -O-甲基转移酶产生。 Cap2 structure and the structure can be produced using Cap3 additional 2 '-O- methyltransferase. 体外转录的mRNA产物进行琼脂糖电泳并显像。 in vitro transcribed mRNA product was subjected to agarose electrophoresis and imaging. 修饰的RNA用Ambion/AppliedBiosystems (Austin, TX)MEGAClear RNA™纯化试剂盒纯化。 RNA was purified by a modified Ambion / AppliedBiosystems (Austin, TX) ™ RNA purification kit the MEGAclear. 利用PureLink™PCR纯化试剂盒(Invitrogen, Carlsbad, CA)进行PCR。 PCR was performed using PureLink ™ PCR Purification Kit (Invitrogen, Carlsbad, CA). 产物在Nanodrop™UV Absorbance (ThermoFisher,Waltham,MA)上定量。 The product in quantitative Nanodrop ™ UV Absorbance (ThermoFisher, Waltham, MA). 产物的质量、紫外吸收性质和可视化在1.2%琼脂糖凝胶上进行。 Quality of the product, and the UV-absorbing properties for visualization on a 1.2% agarose gel. 产物在TE缓冲液中重悬。 The product was resuspended in TE buffer.

[0199] 示例性的加帽结构可以在体外转录反应期间,按照厂家说明利用用以产生5'鸟苷帽子结构的以下化学RNA帽子类似物同时完成修饰RNA的5'加帽:3' -0-Me_m7G(5')ppp (5,) G ;G (5,) ppp (5,) A ;G (5,) ppp (5,) G ;m7G(5, ) ppp (5,) A ;m7G(5, ) ppp (5,) G (NewEngland BioLabs, Ipswich,MA)。 [0199] Exemplary capping structure may be performed during in vitro transcription reaction, according to the manufacturer's instructions using a 5 'RNA cap analogs following Chemical guanosine cap structure while the complete modified RNA' to generate a capped 5: 3 '-0 -Me_m7G (5 ') ppp (5,) G; G (5,) ppp (5,) A; G (5,) ppp (5,) G; m7G (5,) ppp (5,) A; m7G (5,) ppp (5,) G (NewEngland BioLabs, Ipswich, MA). 可以利用用以产生“CapO”结构(m7G(5,)ppp (5,)G)的痘病毒加帽酶在转录后完成修饰RNA的5'加帽(NewEngland BioLabs, Ipswich,MA)。 Can be used to generate "CapO" structure (m7G (5,) ppp (5,) G) is completed poxvirus capping enzyme modified RNA 5 'capping (NewEngland BioLabs, Ipswich, MA) after transcription. 可以利用痘病毒加帽酶和2' -氧-甲基转移酶产生Capl结构(m7G(5')ppp(5')G-2' -O-甲基)。 May be utilized poxvirus capping enzyme, and 2 '- O - methyl transferase enzyme produced Capl structure (m7G (5') ppp (5 ') G-2' -O- methyl). 可以通过2' -氧-甲基转移酶将5'端倒数第三个核苷酸2' -氧-甲基化从而自Capl结构产生Cap2结构。 By 2 '- O - methyl transferases 5' end of the nucleotide antepenultimate 2 '- O - methyl Cap2 of producing structures from Capl structure. 可以通过2' -氧-甲基转移酶将5' -倒数第二个核苷酸2' -氧-甲基化从而自Cap 2结构产生Cap 3结构。 2 can penultimate nucleotide 2 '- - oxo -' - oxo --5 methyltransferase enzyme 'methylation thereby resulting structure 3 from the structure 2 of Cap Cap. 酶优选获自于重组来源。 The enzyme is preferably obtained from recombinant sources.

[0200] 当转染哺乳动物细胞时,修饰的mRNA可在12_18小时之内保持稳定或保持稳定的时间大于18小时(例如,24、36、48、60、72小时或者高于72小时)。 [0200] When the transfection of mammalian cells, modified mRNA may be stable for hours or 12_18 remain stable for longer than 18 hours (e.g., 24,36,48,60,72 hours, or higher than 72 hours).

[0201] 实施例2:在模式生物反应器中表达作为治疗药物的人G-CSF的哺乳动物商业生产细胞系的再生 [0201] Example 2: the expression pattern in a bioreactor as a human therapeutic agent regeneration mammalian G-CSF commercial production cell lines

[0202] 人粒细胞集落刺激因子(G-CSF)前体的核酸序列如SEQ ID NO:1所示: [0202] human granulocyte colony stimulating factor is a nucleic acid sequence of the precursor (G-CSF) as SEQ ID NO: 1 shown in:

[0203] agcttttggaccctcgtacagaaa`(SEQIDN0:1) [0203] agcttttggaccctcgtacagaaa` (SEQIDN0: 1)

[0204] G-CSF mRNA 的核酸序列如SEQ ID NO:2 所示: [0204] G-CSF mRNA nucleic acid sequence as SEQ ID NO: 2 below:

[0205] (SEQIDNO:2) [0205] (SEQIDNO: 2)

[0206] 示例性G-CSF修饰mRNA (modRNA)的核酸序列如SEQ ID N0:3所示: [0206] Exemplary modified G-CSF mRNA (modRNA) a nucleic acid sequence as SEQ ID N0: Figure 3:

[0207] ag5meCψ ψ ψ ψ gga5meC5meC5meCΨ5meCgΨa5meCagaag5meCΨ aa Ψa5meCga5meCΨ5meCa5meCΨ a ΨagggaaaΨaagagagaaaagaagagΨaagaagaaaΨ a Ψ aagag5meC5meCa5meC5meCaΨgg5meC5meCggΨ5meC5meC5meCg5meCga5meC5meC5meCaaag5meC5meC5meC5meCaΨgaaa5meCΨ Ψ a Ψ gg5meC5meC5meCΨg5meCagΨ Ψg5meCΨg5meCψ ψ ψ gg5meCa5meCΨ5meCgg5meC5meC5meCΨ5meCΨgga5meCagΨ5meC5meCaagaag5meCga5meCΨ5meC5meC Ψ5meCΨ5meCgga5meC5meCΨg5meC5meCΨ5meCaΨ5meCgΨ Ψg5meC5meCg5meCagΨ5meCaΨ Ψ5meC5meCΨΨ Ψ ΨgaagΨ g Ψ5meCΨggag5meCaggΨg5meCgaaagaΨ Ψ5meCaggg5meCgaΨggag5meC5meCg5meCa5meCΨ5meC5meCaagagaag5meCΨ5meCΨg5meCg5meCga5meCa Ψa5meCaaa5meCΨ Ψ Ψg5meC5meCaΨ5meC5meC5meCgaggag5meCΨ5meCg Ψa5meC Ψg5meC Ψ5meCggg5meCa5meCag5meC Ψ ΨggggaΨ Ψ5meC5meC5meCΨggg5meC Ψ5meC5meC Ψ5meC Ψ5meC Ψ5meCgΨ5meC5meCΨg Ψ 5meC5meCg Ψ 5meCg5meCagg5meC ψ ψ ψ g5meCag Ψ Ψ gg5meCaggg Ψ g5meC5meC ψ ψ ψ 5meC5meC5meCag5meC Ψ 5meC5meCa5meC Ψ 5meC5meCgg ¥ ¥ ¥ g ¥ ¥ 5meC Ψ Ψ g Ψ a Ψ 5meCaggga5meC [0207] ag5meCψ ψ ψ ψ gga5meC5meC5meCΨ5meCgΨa5meCagaag5meCΨ aa Ψa5meCga5meCΨ5meCa5meCΨ a ΨagggaaaΨaagagagaaaagaagagΨaagaagaaaΨ a Ψ aagag5meC5meCa5meC5meCaΨgg5meC5meCggΨ5meC5meC5meCg5meCga5meC5meC5meCaaag5meC5meC5meC5meCaΨgaaa5meCΨ Ψ a Ψ gg5meC5meC5meCΨg5meCagΨ Ψg5meCΨg5meCψ ψ ψ gg5meCa5meCΨ5meCgg5meC5meC5meCΨ5meCΨgga5meCagΨ5meC5meCaagaag5meCga5meCΨ5meC5meC Ψ5meCΨ5meCgga5meC5meCΨg5meC5meCΨ5meCaΨ5meCgΨ Ψg5meC5meCg5meCagΨ5meCaΨ Ψ5meC5meCΨΨ Ψ ΨgaagΨ g Ψ5meCΨggag5meCaggΨg5meCgaaagaΨ Ψ5meCaggg5meCgaΨggag5meC5meCg5meCa5meCΨ5meC5meCaagagaag5meCΨ5meCΨg5meCg5meCga5meCa Ψa5meCaaa5meCΨ Ψ Ψg5meC5meCaΨ5meC5meC5meCgaggag5meCΨ5meCg Ψa5meC Ψg5meC Ψ5meCggg5meCa5meCag5meC Ψ ΨggggaΨ Ψ5meC5meC5meCΨggg5meC Ψ5meC5meC Ψ5meC Ψ5meC Ψ5meCgΨ5meC5meCΨg Ψ 5meC5meCg Ψ 5meCg5meCagg5meC ψ ψ ψ g5meCag Ψ Ψ gg5meCaggg Ψ g5meC5meC ψ ψ ψ 5meC5meC5meCag5meC Ψ 5meC5meCa5meC Ψ 5meC5meCgg ¥ ¥ ¥ g ¥ ¥ 5meC Ψ Ψ g Ψ a Ψ 5meCaggga5meC Ψg5meCΨg5meCaag5meC5meC5meCΨ ΨgagggaaΨ5meCΨ5meCg5meC5meCagaaΨ Ψggg5meC5meC5meCga5meCg5meCΨgga5meCa5meCgΨ Ψg5meCag5meCΨ5meCga5meCgΨgg5meCggaΨΨ Ψ5meCg5meCaa5meCaa5meC5meCaΨ5meC Ψgg5meCag5meCagaΨggaggaa5meCΨggggaΨgg5meCa5meC5meC5meCg5meCg5meCΨg5meCag5meC5meC5meCa5meCg5meCaggggg5meCaaΨg5meC5meCgg5meC5meC Ψ Ψ Ψg5meCg Ψ5meC5meCg5meCg Ψ Ψ Ψ5meCag5meCg5meCaggg5meCggg ΨggagΨ5meC5meCΨ5meCgΨag5meCgag5meC5meCa5meC5meCΨ Ψ5meCaaΨ5meCaΨ Ψ Ψ Ψ Ψ ggaagΨ5meCΨ5meCg Ψa5meC5me CgggΨg5meC Ψgaga5meCa Ψ5meC Ψ Ψg5meCg5meCag5meC5meCgΨgaag5meCg5meC Ψg5meC5meCΨ Ψ5meCΨg5meCgggg5meCΨ Ψg5meC5meCΨ Ψ5meCΨgg5meC5meCaΨg5meC5meC5meCΨ Ψ5meCΨ Ψ5meC Ψ5meC Ψ5meC5meC5meCΨ Ψg5meCa5meC5meCΨ g Ψ a5meC5meC Ψ 5meC ¥ ¥ gg ¥ 5meC ψ ψ ψ gaa Ψ aaag5meC5meC Ψ gag Ψ aggaagg5meCgg5meC5meCg5meCΨ5meCgag5meCaΨg5meCaΨ5meCΨagaggg5meC5meC5meCaaΨ Ψ5meCg5meC5meC5meC Ψ a Ψ Ψ 5meCgaag Ψ5meCg(SEQ ID NO:3) Ψg5meCΨg5meCaag5meC5meC5meCΨ ΨgagggaaΨ5meCΨ5meCg5meC5meCagaaΨ Ψggg5meC5meC5meCga5meCg5meCΨgga5meCa5meCgΨ Ψg5meCag5meCΨ5meCga5meCgΨgg5meCggaΨΨ Ψ5meCg5meCaa5meCaa5meC5meCaΨ5meC Ψgg5meCag5meCagaΨggaggaa5meCΨggggaΨgg5meCa5meC5meC5meCg5meCg5meCΨg5meCag5meC5meC5meCa5meCg5meCaggggg5meCaaΨg5meC5meCgg5meC5meC Ψ Ψ Ψg5meCg Ψ5meC5meCg5meCg Ψ Ψ Ψ5meCag5meCg5meCaggg5meCggg ΨggagΨ5meC5meCΨ5meCgΨag5meCgag5meC5meCa5meC5meCΨ Ψ5meCaaΨ5meCaΨ Ψ Ψ Ψ Ψ ggaagΨ5meCΨ5meCg Ψa5meC5me CgggΨg5meC Ψgaga5meCa Ψ5meC Ψ Ψg5meCg5meCag5meC5meCgΨgaag5meCg5meC Ψg5meC5meCΨ Ψ5meCΨg5meCgggg5meCΨ Ψg5meC5meCΨ Ψ5meCΨgg5meC5meCaΨg5meC5meC5meCΨ Ψ5meCΨ Ψ5meC Ψ5meC Ψ5meC5meC5meCΨ Ψg5meCa5meC5meCΨ g Ψ a5meC5meC Ψ 5meC ¥ ¥ gg ¥ 5meC ψ ψ ψ gaa Ψ aaag5meC5meC Ψ gag Ψ aggaagg5meCgg5meC5meCg5meCΨ5meCgag5meCaΨg5meCaΨ5meCΨagaggg5meC5meC5meCaaΨ Ψ5meCg5meC5meC5meC Ψ a Ψ Ψ 5meCgaag Ψ5meCg (SEQ ID NO: 3)

[0208] 图1显示了获自转染有G-CSF的modRNA的中国仓鼠卵巢细胞(CHO)的人粒细胞集落刺激因子(G-CSF)的酶联免疫试验(ELISA)。 [0208] Figure 1 shows eligible rotation contaminated modRNA G-CSF in Chinese hamster ovary cells (CHO) human granulocyte colony stimulating factor (G-CSF) enzyme-linked immunoassay (ELISA). CHO细胞在补充了L-谷氨酰、次黄嘌呤和胸腺嘧啶核苷的⑶CHO培养基中生长。 CHO cells were supplemented with L- glutamyl, ⑶CHO medium hypoxanthine and thymidine grown. 在盛有7ml培养基的获自Corning的75cm2培养皿中用复合了购自Invitrogen 的RNAiMax 的24 μ g modRNA 转染2X IO6 个细胞。 In the medium filled with 7ml of 75cm2 petri dish available from Corning complexed with commercially available from Invitrogen 24 μ g modRNA RNAiMax of 2X IO6 transfected cells. RNA:RNAiMAX复合体的制备:首先,将RNA在室温下与5X体积稀释的CD CHO培养基孵育10分钟;在第二个瓶中,将RNAiMAX试剂在室温下与10X体积稀释的⑶CHO培养基孵育10分钟;随后将RNA瓶与RNAiMAX瓶混合并在室温下孵育20-30,然后以逐滴方式加入至细胞中。 RNA: RNAiMAX Preparation of composite: First, the RNA was incubated for 10 minutes with 5X volume of diluted CD CHO medium at room temperature; in a second vial, RNAiMAX reagent diluted at room temperature with a 10X volume of medium incubated ⑶CHO 10 minutes; RNA bottle was then mixed and incubated 20-30 RNAiMAX bottle at room temperature and then added in a dropwise manner to the cells. 在转染后12和24小时测量培养基中分泌的huG-CSF浓度。 Secretion at 12 and 24 hours after transfection the medium was measured huG-CSF concentrations. 将细胞悬液贮存在_20°C。 The cell suspension was stored at _20 ° C. 按厂家说明使用购自Invitrogen的ELISA试剂盒测定自转染的人胚肾细胞中分泌的人粒细胞集落刺激因子(G-CSF)的量。 According to the manufacturer's instructions using the commercially available ELISA kit from Invitrogen assay the transfected human embryonic kidney cells secreted human granulocyte colony stimulating amount of factor (G-CSF) is. 这些数据显示,huG-CSF modRNA (SEQ ID NO: 3)能在CHO细胞中翻译,huG-CSF被分泌到细胞外并释放到细胞外环境中。 These data show that, huG-CSF modRNA (SEQ ID NO: 3) can be translated in CHO cells, huG-CSF is secreted outside the cell and released into the extracellular environment. 进一步,这些数据证明为生产分泌的蛋白质的目的,用modRNA huG-CSF对细胞进行转染而进行分泌蛋白的生产规模可以等比例放大生物反应器或大型细胞培养条件。 Further, these data demonstrate that for the purpose of production of secreted proteins, with modRNA huG-CSF Cells were transfected to scale production of secreted proteins and the like can be scaled larger or bioreactor cell culture conditions.

[0209]实施例3:表达作为治疗药剂的人源化IgG抗体(曲妥珠单抗和利妥昔单抗)的哺乳动物商业生产细胞株在模式生物反应器中的再生(de novo) [0209] Example 3: As the expression of human therapeutic agents regeneration humanized IgG antibody (trastuzumab and rituximab) commercial production of mammalian cell lines in the pattern of the bioreactor (de novo)

[0210] 利妥昔单抗重链的核酸序列如SEQ ID N0:4所示: [0210] rituximab nucleic acid sequences of the heavy chain such as SEQ ID N0: 4 shown:

[0211 ] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGATGGCGCCGAGGACCCTGGTGCTCTTGCTCACGGGTGCCTTGGCCCTCACGCAAACATGGGCGGGACAGGCGTACTTGCAGCAGTCAGGGGCAGAACTCGTAAGGCCCGGAGCGTCGGTGAAGATGTCGTGTAAAGCGTCGGGCTATACTTTCACATCGTACAACATGCACTGGGTCAAACAGACGCCCCGACAAGGGCTGGAGTGGATTGGAGCTATCTACCCCGGTAACGGGGATACGTCGTACAACCAGAAGTTTAAGGGGAAGGCGACTCTTACTGTCGACAAGTCGTCCTCCACCGCCTATATGCAGCTGTCGAGCCTGACTTCGGAAGATTCAGCGGTGTACTTTTGTGCGCGCGTGGTCTATTACTCAAATTCGTATTGGTATTTCGATGTGTGGGGTACGGGGACCACTGTGACCGTGTCAGGACCCTCGGTATTCCCCCTCGCGCCTAGCTCAAAGTCCACCTCCGGGGGAACAGCCGCCTTGGGTTGCTTGGTAAAGGACTATTTCCCCGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCACTGACATCGGGAGTGCACACGTTTCCCGCGGTACTTCAGTCATCAGGACTCTACTCGCTGTCAAGCGTGGTCACGGTGCCTTCATCCTCCCTTGGAACGCAGACTTACATCTGCAACGTGAATCATAAGCCTAGCAATACCAAGGTCGACAAGAAAGCCGAACCCAAATCATGTGATAAAACACACACGTGTCCTCCCTGCCCCGCACCGGAGCTTCTCGGGGGACCGAGCGTGTTCTTGTTTCCACCTAAGCCGAAAGATACGCTTATGATCTCCCGGACCCCCGAAGTAACTTGCGTAGTAGTAGACGTAAGCCACGAGGACCCCGAAGTGAAATTCAATTGGTACGTCGAC [0211] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGATGGCGCCGAGGACCCTGGTGCTCTTGCTCACGGGTGCCTTGGCCCTCACGCAAACATGGGCGGGACAGGCGTACTTGCAGCAGTCAGGGGCAGAACTCGTAAGGCCCGGAGCGTCGGTGAAGATGTCGTGTAAAGCGTCGGGCTATACTTTCACATCGTACAACATGCACTGGGTCAAACAGACGCCCCGACAAGGGCTGGAGTGGATTGGAGCTATCTACCCCGGTAACGGGGATACGTCGTACAACCAGAAGTTTAAGGGGAAGGCGACTCTTACTGTCGACAAGTCGTCCTCCACCGCCTATATGCAGCTGTCGAGCCTGACTTCGGAAGATTCAGCGGTGTACTTTTGTGCGCGCGTGGTCTATTACTCAAATTCGTATTGGTATTTCGATGTGTGGGGTACGGGGACCACTGTGACCGTGTCAGGACCCTCGGTATTCCCCCTCGCGCCTAGCTCAAAGTCCACCTCCGGGGGAACAGCCGCCTTGGGTTGCTTGGTAAAGGACTATTTCCCCGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCACTGACATCGGGAGTGCACACGTTTCCCGCGGTACTTCAGTCATCAGGACTCTACTCGCTGTCAAGCGTGGTCACGGTGCCTTCATCCTCCCTTGGAACGCAGACTTACATCTGCAACGTGAATCATAAGCCTAGCAATACCAAGGTCGACAAGAAAGCCGAACCCAAATCATGTGATAAAACACACACGTGTCCTCCCTGCCCCGCACCGGAGCTTCTCGGGGGACCGAGCGTGTTCTTGTTTCCACCTAAGCCGAAAGATACGCTTATGATCTCCCGGACCCCCGAAGTAACTTGCGTAGTAGTAGACGTAAGCCACGAGGACCCCGAAGTGAAATTCAATTGGTACGTCGAC GGAGTGGAGGTCCATAATGCGAAAACAAAGCCGAGAGAGGAACAGTACAATTCCACATACCGCGTCGTAAGCGTCTTGACAGTATTGCATCAGGATTGGCTGAACGGAAAGGAATACAAGTGCAAAGTATCAAACAAAGCACTTCCGGCACCGATTGAAAAGACGATCTCAAAAGCAAAA GGGCAACCTCGGGAGCCACAAGTCTATACTCTCCCGCCGTCGCGCGATGAATTGACCAAAAACCAGGTGTCCCTTACATGTCTCGTAAAGGGTTTTTACCCGTCAGACATCGCCGTCGAGTGGGAGTCAAACGGTCAGCCGGAGAATAACTATAAGACGACCCCACCAGTCTTGGACAGCGATGGCTCCTTCTTCTTGTATTCAAAGCTGACGGTGGACAAATCGAGATGGCAGCAGGGTAATGTGTTTTCGTGCAGCGTCATGCACGAGGCGCTTCATAATCATTACACTCAAAAGTCCCTGTCGCTGTCGCCCGGAAAGCACCATCACCACCACCATTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA (SEQ ID NO:4) GGAGTGGAGGTCCATAATGCGAAAACAAAGCCGAGAGAGGAACAGTACAATTCCACATACCGCGTCGTAAGCGTCTTGACAGTATTGCATCAGGATTGGCTGAACGGAAAGGAATACAAGTGCAAAGTATCAAACAAAGCACTTCCGGCACCGATTGAAAAGACGATCTCAAAAGCAAAA GGGCAACCTCGGGAGCCACAAGTCTATACTCTCCCGCCGTCGCGCGATGAATTGACCAAAAACCAGGTGTCCCTTACATGTCTCGTAAAGGGTTTTTACCCGTCAGACATCGCCGTCGAGTGGGAGTCAAACGGTCAGCCGGAGAATAACTATAAGACGACCCCACCAGTCTTGGACAGCGATGGCTCCTTCTTCTTGTATTCAAAGCTGACGGTGGACAAATCGAGATGGCAGCAGGGTAATGTGTTTTCGTGCAGCGTCATGCACGAGGCGCTTCATAATCATTACACTCAAAAGTCCCTGTCGCTGTCGCCCGGAAAGCACCATCACCACCACCATTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA (SEQ ID NO: 4)

[0212] 利妥昔单抗重链的mRNA的核酸序列如SEQ ID NO: 5所示: [0212] Rituximab heavy chain nucleic acid sequence mRNA, such as SEQ ID NO: 5 shown:

[0213] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGAGGACCCUGGUGCUCUUGCUCACGGGUGCCUUGGCCCUCACGCAAACAUGGGCGGGACAGGCGUACUUGCAGCAGUCAGGGGCAGAACUCGUAAGGCCCGGAGCGUCGGUGAAGAUGUCGUGUAAAGC⑶CGGGCUAUACUUUCACAUCGUACAACAUGCACUGGGUCAAACAGACGCCCCGACAAGGGCUGGAGUGGAUUGGAGCUAUCUACCCCGGUAACGGGGAUACGUCGUACAACCAGAAGUUUAAGGGGAAGGCGACUCUUACUGUCGACAAGUCGUCCUCCACCGCCUAUAUGCAGCUGUCGAGCCUGACUUCGGAAGAUUCAGCGGUGUACUUUUGUGCGCGCGUGGUCUAUUACUCAAAUUCGUAUUGGUAUUUCGAUGUGUGGGGUACGGGGACCACUGUGACCGUGUCAGGACCCUCGGUAUUCCCCCUCGCGCCUAGCUCAAAGUCCACCUCCGGGGGAACAGCCGCCUUGGGUUGCUUGGUAAAGGACUAUUUCCCCGAGCCC⑶腳CUCGUAAAGGGUUUUUACCCGUCAGACAUCGCCGUCGAGUGGGAGUCAAACGGUCAGCCGGAGAAUAACUAUAAGACGACCCCACCA⑶CUUGGACAGCGAUGGCUCCUUCUUCUUGUAUUCAAAGCUGACGGUGGACAAAUCGAGAUGGCAGCAGGGUAAUGUGUUUUCGUGCAGCGUCAUGCACGAGGCGCUUCAUAAUCAUUACACUCAAAAGUCCCUGUCGC腳CGCCCGGAAAGCACCAUCACCACCACCAUUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUG [0213] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGAGGACCCUGGUGCUCUUGCUCACGGGUGCCUUGGCCCUCACGCAAACAUGGGCGGGACAGGCGUACUUGCAGCAGUCAGGGGCAGAACUCGUAAGGCCCGGAGCGUCGGUGAAGAUGUCGUGUAAAGC⑶CGGGCUAUACUUUCACAUCGUACAACAUGCACUGGGUCAAACAGACGCCCCGACAAGGGCUGGAGUGGAUUGGAGCUAUCUACCCCGGUAACGGGGAUACGUCGUACAACCAGAAGUUUAAGGGGAAGGCGACUCUUACUGUCGACAAGUCGUCCUCCACCGCCUAUAUGCAGCUGUCGAGCCUGACUUCGGAAGAUUCAGCGGUGUACUUUUGUGCGCGCGUGGUCUAUUACUCAAAUUCGUAUUGGUAUUUCGAUGUGUGGGGUACGGGGACCACUGUGACCGUGUCAGGACCCUCGGUAUUCCCCCUCGCGCCUAGCUCAAAGUCCACCUCCGGGGGAACAGCCGCCUUGGGUUGCUUGGUAAAGGACUAUUUCCCCGAGCCC⑶ foot CUCGUAAAGGGUUUUUACCCGUCAGACAUCGCCGUCGAGUGGGAGUCAAACGGUCAGCCGGAGAAUAACUAUAAGACGACCCCACCA⑶CUUGGACAGCGAUGGCUCCUUCUUCUUGUAUUCAAAGCUGACGGUGGACAAAUCGAGAUGGCAGCAGGGUAAUGUGUUUUCGUGCAGCGUCAUGCACGAGGCGCUUCAUAAUCAUUACACUCAAAAGUCCCUGUCGC foot CGCCCGGAAAGCACCAUCACCACCACCAUUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUG CAUCUAGA(SEQ ID NO:5) CAUCUAGA (SEQ ID NO: 5)

[0214] 利妥昔单抗轻链的核酸序列的核酸序列如SEQ ID NO:6所示: [0214] nucleic acid sequence of the light chain of rituximab as SEQ ID NO: 6 as shown:

[0215] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGTCATGGCCCCGAGAACACTTGTGCTGTTGTTGACAGGAGCGCTCGCACTCACACAGACTTGGGCCGGTCAGATTGTGCTCAGCCAGTCGCCAGCGATCCTTTCGGCCTCCCCTGGTGAGAAAGTAACGATGACGTGCCGAGCCTCCTCAAGCGTGTCATACATGCATTGGTATCAGCAGAAGCCTGGGTCGTCGCCCAAGCCCTGGATCTACGCCCCGTCCAATCTTGCGTCAGGGGTCCCGGCACGGTTCAGCGGATCGGGGTCGGGTACATCGTATTCACTCACGATTAGCCGCGTAGAGGCCGAGGACGCGGCGACTTACTACTGTCAGCAATGGTCCTTTAATCCACCCACGTTTGGAGCGGGCACCAAGCTCGAACTTAAAAGAACGGTCGCCGCACCCTCAGTGTTTATCTTCCCGCCCTCGGACGAACAACTTAAGTCGGGGACCGCTTCCGTGGTGTGCTTGCTGAACAATTTCTATCCTCGGGAAGCTAAAGTGCAATGGAAAGTCGATAACGCATTGCAGAGCGGAAACTCACAAGAGTCGGTAACTGAGCAGGATAGCAAGGATTCGACATACTCGCTGAGCAGCACGCTGACGTTGTCCAAGGCGGACTACGAGAAACACAAGGTATATGCGTGTGAAGTCACCCACCAGGGATTGTCATCGCCGGTCACCAAATCATTCAACA GGTGATAAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA(SEQ IDNO: 6) [0215] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGTCATGGCCCCGAGAACACTTGTGCTGTTGTTGACAGGAGCGCTCGCACTCACACAGACTTGGGCCGGTCAGATTGTGCTCAGCCAGTCGCCAGCGATCCTTTCGGCCTCCCCTGGTGAGAAAGTAACGATGACGTGCCGAGCCTCCTCAAGCGTGTCATACATGCATTGGTATCAGCAGAAGCCTGGGTCGTCGCCCAAGCCCTGGATCTACGCCCCGTCCAATCTTGCGTCAGGGGTCCCGGCACGGTTCAGCGGATCGGGGTCGGGTACATCGTATTCACTCACGATTAGCCGCGTAGAGGCCGAGGACGCGGCGACTTACTACTGTCAGCAATGGTCCTTTAATCCACCCACGTTTGGAGCGGGCACCAAGCTCGAACTTAAAAGAACGGTCGCCGCACCCTCAGTGTTTATCTTCCCGCCCTCGGACGAACAACTTAAGTCGGGGACCGCTTCCGTGGTGTGCTTGCTGAACAATTTCTATCCTCGGGAAGCTAAAGTGCAATGGAAAGTCGATAACGCATTGCAGAGCGGAAACTCACAAGAGTCGGTAACTGAGCAGGATAGCAAGGATTCGACATACTCGCTGAGCAGCACGCTGACGTTGTCCAAGGCGGACTACGAGAAACACAAGGTATATGCGTGTGAAGTCACCCACCAGGGATTGTCATCGCCGGTCACCAAATCATTCAACA GGTGATAAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA (SEQ IDNO: 6)

[0216] 利妥昔单抗的轻链的mRNA的核酸序列如SEQ ID NO: 7所示。 [0216] nucleic acid sequences of the light chain mRNA rituximab as SEQ ID NO: 7 shown in FIG. CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCUGUCAUGGCCCCGAGAACACUUGUGCUGUUGUUGACAGGAGCGCUCGCACUCACACAGACUUGGGCCGGUCAGAUUGUGCUCAGCCAGUCGCCAGCGAUCCUUUCGGCCUCCCCUGGUGAGAAAGUAACGAUGACGUGCCGAGCCUCCUCAAGCGUGUCAUACAUGCAUUGGUAUCAGCAGAAGCCUGGGUCGUCGCCCAAGCCCUGGAUCUACGCCCCGUCCAAUCUUGCGUCAGGGGUCCCGGCACGGUUCAGCGGAUCGGG⑶CGGGUACAUCGUAUUCACUCACGAUUAGCCGCGUAGAGGCCGAGGACGCGGCGACUUACUACUGUCAGCAAUGGUCCUUUAAUCCACCCACGUUUGGAGCGGGCACCAAGCUCGAACUUAAAAGAACGGUCGCCGCACCCUCAGUGUUUAUCUUCCCGCCCUCGGACGAACAACUUAAGUCGGGGACCGCUUCCGUGGUGUGCUUGCUGAACAAUUUCUAUCCUCGGGAAGCUAAAGUGCAAUGGAAAGUCGAUAACGCAUUGCAGAGCGGAAACUCACAAGAGUCG ⑶ AA ⑶ GAGCAGGAUAGCAAGGAUUCGACAUA ⑶ CG ⑶ GAGCAGCACG ⑶ GAC ⑶腳CCAAGGCGG^^AAACACAAGGUAUAUGCGUGUGAAGUCACCCACCAGGGAUUGUCAUCGCCGGUCACCAAAUCAUUCAACAGGUGAUAAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA(SEQ ID NO:7) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCUGUCAUGGCCCCGAGAACACUUGUGCUGUUGUUGACAGGAGCGCUCGCACUCACACAGACUUGGGCCGGUCAGAUUGUGCUCAGCCAGUCGCCAGCGAUCCUUUCGGCCUCCCCUGGUGAGAAAGUAACGAUGACGUGCCGAGCCUCCUCAAGCGUGUCAUACAUGCAUUGGUAUCAGCAGAAGCCUGGGUCGUCGCCCAAGCCCUGGAUCUACGCCCCGUCCAAUCUUGCGUCAGGGGUCCCGGCACGGUUCAGCGGAUCGGG⑶CGGGUACAUCGUAUUCACUCACGAUUAGCCGCGUAGAGGCCGAGGACGCGGCGACUUACUACUGUCAGCAAUGGUCCUUUAAUCCACCCACGUUUGGAGCGGGCACCAAGCUCGAACUUAAAAGAACGGUCGCCGCACCCUCAGUGUUUAUCUUCCCGCCCUCGGACGAACAACUUAAGUCGGGGACCGCUUCCGUGGUGUGCUUGCUGAACAAUUUCUAUCCUCGGGAAGCUAAAGUGCAAUGGAAAGUCGAUAACGCAUUGCAGAGCGGAAACUCACAAGAGUCG ⑶ AA ⑶ GAGCAGGAUAGCAAGGAUUCGACAUA ⑶ CG ⑶ GAGCAGCACG ⑶ GAC ⑶ foot CCAAGGCGG ^^ AAACACAAGGUAUAUGCGUGUGAAGUCACCCACCAGGGAUUGUCAUCGCCGGUCACCAAAUCAUUCAACAGGUGAUAAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA (SEQ ID NO: 7)

[0217] 曲妥珠单抗(Trastuzumab)的重链的核酸序列的核酸序列如SEQ ID N0:8所示: [0217] Trastuzumab (of Trastuzumab) a nucleic acid sequence of the heavy chain such as SEQ ID N0: 8 shown:

[0218] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGATGGCGCCGCGGACCCTGGTCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAGACCTGGGCCGGGGAGGTGCAGCTGGTCGAGAGCGGCGGGGGCCTCGTGCAGCCGGGCGGGTCGCTGCGGCTGAGCTGCGCCGCGAGCGGGTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTCGAGTGGGTCGCCCGGATCTACCCCACGAACGGGTACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCGGACACCTCGAAGAACACGGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCAGCCGGTGGGGCGGCGACGGGTTCTACGCCATGGACTACTGGGGGCAGGGCACCCTCGTCACCGTGAGCAGCGCGTCGACGAAGGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACAAGACCCACACCTGCCCGCCCTGCCCCGCCCCCGAGCTCC (SEQ ID NO:8) [0218] CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGATGGCGCCGCGGACCCTGGTCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAGACCTGGGCCGGGGAGGTGCAGCTGGTCGAGAGCGGCGGGGGCCTCGTGCAGCCGGGCGGGTCGCTGCGGCTGAGCTGCGCCGCGAGCGGGTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTCGAGTGGGTCGCCCGGATCTACCCCACGAACGGGTACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCGGACACCTCGAAGAACACGGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCAGCCGGTGGGGCGGCGACGGGTTCTACGCCATGGACTACTGGGGGCAGGGCACCCTCGTCACCGTGAGCAGCGCGTCGACGAAGGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACAAGACCCACACCTGCCCGCCCTGCCCCGCCCCCGAGCTCC (SEQ ID NO: 8)

[0219] 曲妥珠单抗的重链的mRNA的核酸序列如SEQ ID NO:9所示: [0219] nucleic acid sequences of the heavy chain mRNA trastuzumab as SEQ ID NO: 9 shown:

[0220] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUG⑶CCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAGACCUGGGCCGGGGAGGUGCAGCUGGUCGAGAGCGGCGGGGGCCUCGUGCAGCCGGGCGGGUCGCUGCGGCUGAGCUGCGCCGCGAGCGGGUUCAACAUCAAGGACACCUACAUCCACUGGGUGCGCCAGGCCCCCGGCAAGGGCCUCGAGUGGGUCGCCCGGAUCUACCCCACGAACGGGUACACCCGCUACGCCGACAGC⑶GAAGGGCCGGUUCACCAUCAGCGCGGACACCUCGAAGAACACGGCCUACCUGCAGAUGAACAGCCUGCGCGCCGAGGACACCGCCGUGUACUACUGCAGCCGGUGGGGCGGCGACGGGUUCUACGCCAUGGACUACUGGGGGCAGGGCACCCUCGUCACCGUGAGCAGCGCGUCGACGAAGGGGCCCAGCGUGUUCCCGCUGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCUGGGCUGCCUCGUCAAGGACUACUUCCCCGAGCCCGUGACCGUGUCGUGGAACAGCGGCGCGCUGACGAGCGGGGUCCACACCUUCCCGGCCGUGCUGCAGAGCAGCGGCCUCUACUCGCUGAGCAGCGUGGUCACCGUGCCCAGCAGCAGCCUGGGGACCCAGACGUACAUCUGCAACGUGAACCACAAGCCCUCGAACACCAAG⑶CGACAAGAAGGUGGAGCCCCCGAAGAGCUGCGACAAGACCCACACCUGCCCGCCCUGCCCCGCCCCCGAG(:UCCUGGGCGGGCCCAGCGUGUUCCUGUUCCCGCCCAAGCCCAAGGACACG⑶CAUGAUCAGCCGCACCCCCGAGGUCACCUGCGUGGUG⑶⑶CUUUGAAUAAAGCCUGAGUAGGAAGGCGG [0220] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUG⑶CCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAGACCUGGGCCGGGGAGGUGCAGCUGGUCGAGAGCGGCGGGGGCCUCGUGCAGCCGGGCGGGUCGCUGCGGCUGAGCUGCGCCGCGAGCGGGUUCAACAUCAAGGACACCUACAUCCACUGGGUGCGCCAGGCCCCCGGCAAGGGCCUCGAGUGGGUCGCCCGGAUCUACCCCACGAACGGGUACACCCGCUACGCCGACAGC⑶GAAGGGCCGGUUCACCAUCAGCGCGGACACCUCGAAGAACACGGCCUACCUGCAGAUGAACAGCCUGCGCGCCGAGGACACCGCCGUGUACUACUGCAGCCGGUGGGGCGGCGACGGGUUCUACGCCAUGGACUACUGGGGGCAGGGCACCCUCGUCACCGUGAGCAGCGCGUCGACGAAGGGGCCCAGCGUGUUCCCGCUGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCUGGGCUGCCUCGUCAAGGACUACUUCCCCGAGCCCGUGACCGUGUCGUGGAACAGCGGCGCGCUGACGAGCGGGGUCCACACCUUCCCGGCCGUGCUGCAGAGCAGCGGCCUCUACUCGCUGAGCAGCGUGGUCACCGUGCCCAGCAGCAGCCUGGGGACCCAGACGUACAUCUGCAACGUGAACCACAAGCCCUCGAACACCAAG⑶CGACAAGAAGGUGGAGCCCCCGAAGAGCUGCGACAAGACCCACACCUGCCCGCCCUGCCCCGCCCCCGAG (: UCCUGGGCGGGCCCAGCGUGUUCCUGUUCCCGCCCAAGCCCAAGGACACG⑶CAUGAUCAGCCGCACCCCCGAGGUCACCUGCGUGGUG⑶⑶CUUUGAAUAAAGCCUGAGUAGGAAGGCGG CCGCUCGAGCAUGCAUCUAGA(SEQ ID NO:9) CCGCUCGAGCAUGCAUCUAGA (SEQ ID NO: 9)

[0221] 曲妥珠单抗轻链的核酸序列的核酸序列如SEQ ID NO: 10所示: [0221] The nucleic acid sequences of trastuzumab light chain such as SEQ ID NO: 10 below:

[0222] (SEQ ID NO:10) [0222] (SEQ ID NO: 10)

[0223] 曲妥珠单抗轻链的mRNA的核酸序列如SEQ ID NO: 11所示: [0223] The nucleic acid sequence mRNA trastuzumab light chain such as SEQ ID NO: 11 shown:

[0224] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUG⑶CCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAGACCUGGGCCGGGGACAUCCAGAUGACCCAGAGCCC⑶CGAGCCUGAGCGCCAGC⑶GGGCGACCGG⑶CACGAUCACCUGCCGCGCGAGCCAGGACGUGAACACCGCC⑶GGCCUGGUACCAGCAGAAGCCCGGGAAGGCCCCCAAGCUCCUGAUCUACUCGGCGAGCUUCCUGUACAGCGGCGUCCCCAGCCGGUUCAGCGG⑶CGCGCAGCGGCACCGACUUCACGCUCACCAUCAGCAGCCUGCAGCCGGAGGACUUCGCCACCUACUACUGCCAGCAGCACUACACCACGCCCCCCACCUUCGGGCAGGGCACCAAGGUGGAGAUCAAGCGGACCGUGGCCGCCCCCAGC⑶CUUCAUCUUCCCGCCCAGCGACGAGCAGCUGAA⑶CGGGCACGGCCAGC⑶GGUGUGCCUCCUGAACAACUUCUACCCCCGCGAGGCGAAG⑶CCA⑶GGAAGGUGGACAACGCCCUGCAGAGCGGGAACAGCCAGGAGAGC⑶GACCGAGCAGGACUCGAAGGACAGCACCUACAGCCUCAGCAGCACCCUGACGCUGAGCAAGGCCGACUACGAGAAGCACAAGGUCUACGCCUGCGAG⑶GACCCACCAGGGGCUCUCGAGCCCCGUGACCAAGAGCUUCAACCGGGGCGA⑶GCUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA(SEQ ID NO:11) [0224] CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUG⑶CCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAGACCUGGGCCGGGGACAUCCAGAUGACCCAGAGCCC⑶CGAGCCUGAGCGCCAGC⑶GGGCGACCGG⑶CACGAUCACCUGCCGCGCGAGCCAGGACGUGAACACCGCC⑶GGCCUGGUACCAGCAGAAGCCCGGGAAGGCCCCCAAGCUCCUGAUCUACUCGGCGAGCUUCCUGUACAGCGGCGUCCCCAGCCGGUUCAGCGG⑶CGCGCAGCGGCACCGACUUCACGCUCACCAUCAGCAGCCUGCAGCCGGAGGACUUCGCCACCUACUACUGCCAGCAGCACUACACCACGCCCCCCACCUUCGGGCAGGGCACCAAGGUGGAGAUCAAGCGGACCGUGGCCGCCCCCAGC⑶CUUCAUCUUCCCGCCCAGCGACGAGCAGCUGAA⑶CGGGCACGGCCAGC⑶GGUGUGCCUCCUGAACAACUUCUACCCCCGCGAGGCGAAG⑶CCA⑶GGAAGGUGGACAACGCCCUGCAGAGCGGGAACAGCCAGGAGAGC⑶GACCGAGCAGGACUCGAAGGACAGCACCUACAGCCUCAGCAGCACCCUGACGCUGAGCAAGGCCGACUACGAGAAGCACAAGGUCUACGCCUGCGAG⑶GACCCACCAGGGGCUCUCGAGCCCCGUGACCAAGAGCUUCAACCGGGGCGA⑶GCUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA (SEQ ID NO: 11)

[0225] 野生型CERT蛋白质的核苷酸序列的核酸序列如SEQ ID NO: 12所示:[0226] at gt cggata atcagagc tg gaactcgtcg ggc t cggagg aggat ccagagacggagtctgggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacattcatgggtggcaggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatctgaagatgaaacagagtatg gctgcagagg atccatctgt cttagcaagg ctgtcatcacacctcacgattttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatcttcgtgctcaggatccagatc atagacagca atggatagat gccattgaac agcacaagactgaatctggatatggatctg aatccagctt gcgtcgacat ggctcaatgg tgtccctggtgtctggagcaagtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagtttacgtgagaagttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacacgctacagaagtactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaagggataaagtggtagaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttcttgcatagtaccaacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaatggtatagactttaaagggg aagcgataac ttttaaagca actactgetg gaatccttgcaacactttctcattgtattg aactaatggt taaacgtgag gacagctggc agaagagactggataaggaaactgag [0225] the nucleotide sequence of a nucleic acid sequence of wild type CERT protein as SEQ ID NO: 12 shown in: [0226] at gt cggata atcagagc tg gaactcgtcg ggc t cggagg aggat ccagagacggagtctgggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacattcatgggtggcaggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatctgaagatgaaacagagtatg gctgcagagg atccatctgt cttagcaagg ctgtcatcacacctcacgattttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatcttcgtgctcaggatccagatc atagacagca atggatagat gccattgaac agcacaagactgaatctggatatggatctg aatccagctt gcgtcgacat ggctcaatgg tgtccctggtgtctggagcaagtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagtttacgtgagaagttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacacgctacagaagtactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaagggataaagtggtagaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttcttgcatagtaccaacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaatggtatagactttaaagggg aagcgataac ttttaaagca actactgetg gaatccttgcaacactttctcattgtattg aactaatggt taaacgtgag gacagctggc agaagagactggataaggaaactgag aaga aaagaagaac agaggaagca tataaaaatg caatgacagaacttaagaaaaaatcccact ttggaggacc agattatgaa gaaggcccta acagtctgattaatgaagaagagttctttg atgctgttga agctgctctt gacagacaag ataaaatagaagaacagtcacagagtgaaa aggtgagatt acattggcct acatccttgc cctctggagatgccttttcttctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaaccacatgacttactcattac aggatgtagg cggagatgcc aattggcagt tggttgtagaagaaggagaaatgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcctttaaaagctacccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctggaatgttgacgttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaacattagctgataatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctcagcgagacgtattatatcttt ctgtcatteg aaagatacca gccttgactg aaaatgaccctgaaacttggatagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccgatgtgtccgtgccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccagagggaaaccaggaaattagca gggacaacat tctatgcaag attacatatg tagctaatgtgaaccctggaggatgggcac cagcctca`gt gttaagggca gtggcaaagc gagagtatcctaaatttctaaaacgtttta cttcttacgt ccaagaaaaa actgcaggaa a aaga aaagaagaac agaggaagca tataaaaatg caatgacagaacttaagaaaaaatcccact ttggaggacc acagtctgattaatgaagaagagttctttg agattatgaa gaaggcccta atttttctgt ggatcatgac agtgctcctc atgctgttga agctgctctt gacagacaag ataaaatagaagaacagtcacagagtgaaa aggtgagatt acattggcct acatccttgc cctctggagatgccttttcttctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaaccacatgacttactcattac aggatgtagg cggagatgcc aattggcagt tggttgtagaagaaggagaaatgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcctttaaaagctacccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctggaatgttgacgttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaacattagctgataatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctcagcgagacgtattatatcttt ctgtcatteg aaagatacca gccttgactg aaaatgaccctgaaacttggatagtttgta taaacaaccgatgtgtccgtgccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccagagggaaaccaggaaattagca gggacaacat tctatgcaag attacatatg tagctaatgtgaaccctggaggatgggcac cagcctca`gt gttaagggca gtggcaaagc gagagtatcctaaatttctaaaacgtttta cttcttacgt ccaagaaaaa actgcaggaa a gcctatttt gttctag (SEQID NO:12) gcctatttt gttctag (SEQID NO: 12)

[0227] 野生型CERT蛋白质的蛋白质序列如SEQ ID NO: 13所示: [0227] Protein sequence of wild type CERT protein as SEQ ID NO: 13 below:

[0228] Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp ProGlu ThrGlu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser LysTrp Thr Asn Tyr lie HisGly Trp Gin Asp Arg Trp Val Val Leu LysAsn Asn Ala Leu Ser Tyr Tyr Lys Ser GluAsp Glu Thr Glu Tyr GlyCys Arg Gly Ser lie Cys Leu Ser Lys Ala Val lie Thr ProHis AspPhe Asp Glu Cys Arg Phe Asp l`ie Ser Val Asn Asp Ser Val Trp TyrLeu ArgAla Gin Asp Pro Asp His Arg Gin Gin Trp lie Asp Ala IleGlu Gin His Lys Thr GluSer Gly Tyr Gly Ser Glu Ser Ser Leu ArgArg His Gly Ser Met Val Ser Leu Val SerGly Ala Ser Gly Tyr SerAla Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu ArgGlu LysLeu Ala Glu Met Glu Thr Phe Arg Asp lie Leu Cys Arg Gin Val AspThr LeuGin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys AspGlu Leu Gin Arg Asp LysVal Val Glu Asp Asp Glu Asp Asp Phe ProThr Thr Arg Ser Asp Gly Asp Phe Leu HisSer Thr Asn Gly Asn LysGlu Lys Leu Phe Pro His Val Thr Pro Lys Gly lie Asn Glylie AspPhe Lys Gly Glu Ala lie Thr Phe Lys Ala Thr Thr Ala Gly lie [0228] Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp ProGlu ThrGlu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser LysTrp Thr Asn Tyr lie HisGly Trp Gin Asp Arg Trp Val Val Leu LysAsn Asn Ala Leu Ser Tyr Tyr Lys Ser GluAsp Glu Thr Glu Tyr GlyCys Arg Gly Ser lie Cys Leu Ser Lys Ala Val lie Thr ProHis AspPhe Asp Glu Cys Arg Phe Asp l`ie Ser Val Asn Asp Ser Val Trp TyrLeu ArgAla Gin Asp Pro Asp His Arg Gin Gin Trp lie Asp Ala IleGlu Gin His Lys Thr GluSer Gly Tyr Gly Ser Glu Ser Ser Leu ArgArg His Gly Ser Met Val Ser Leu Val SerGly Ala Ser Gly Tyr SerAla Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu ArgGlu LysLeu Ala Glu Met Glu Thr Phe Arg Asp lie Leu Cys Arg Gin Val AspThr LeuGin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys AspGlu Leu Gin Arg Asp LysVal Val Glu Asp Asp Glu Asp Asp Phe ProThr Thr Arg Ser Asp Gly Asp Phe Leu HisSer Thr Asn Gly Asn LysGlu Lys Leu Phe Pro His Val Thr Pro Lys Gly lie Asn Glylie AspPhe Lys Gly Glu Ala lie Thr Phe Lys Ala Thr Thr Ala Gly lie LeuAla ThrLeu Ser His Cys lie Glu Leu Met Val Lys Arg Glu Asp SerTrp Gin Lys Arg Leu AspLys Glu Thr Glu Lys Lys Arg Arg Thr GluGlu Ala Tyr Lys Asn Ala Met Thr Glu LeuLys Lys Lys Ser His PheGly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu lie AsnGlu GluGlu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys IleGlu GluGin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr SerLeu Pro Ser Gly Asp AlaPhe Ser Ser Val Gly Thr His Arg Phe ValGin Lys Val Glu Glu Met Val Gin Asn HisMet Thr Tyr Ser Leu GinAsp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu GluGly GluMet Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly lie Val Leu AspPro LeuLys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu ValCys Asn Tyr Phe Trp AsnVal Asp Val Arg Asn Asp Trp Glu Thr ThrIle Glu Asn Phe His Val Val Glu Thr LeuAla Asp Asn Ala lie IleIle Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin ArgAsp ValLeu Tyr Leu Ser Val lie Arg Lys lie Pro Ala Leu Thr Glu Asn AspPro GluThr Trp lie Val Cys Asn Phe Ser Val Asp His Asp Ser AlaP LeuAla ThrLeu Ser His Cys lie Glu Leu Met Val Lys Arg Glu Asp SerTrp Gin Lys Arg Leu AspLys Glu Thr Glu Lys Lys Arg Arg Thr GluGlu Ala Tyr Lys Asn Ala Met Thr Glu LeuLys Lys Lys Ser His PheGly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu lie AsnGlu GluGlu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys IleGlu GluGin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr SerLeu Pro Ser Gly Asp AlaPhe Ser Ser Val Gly Thr His Arg Phe ValGin Lys Val Glu Glu Met Val Gin Asn HisMet Thr Tyr Ser Leu GinAsp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu GluGly GluMet Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly lie Val Leu AspPro LeuLys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu ValCys Asn Tyr Phe Trp AsnVal Asp Val Arg Asn Asp Trp Glu Thr ThrIle Glu Asn Phe His Val Val Glu Thr LeuAla Asp Asn Ala lie IleIle Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin ArgAsp ValLeu Tyr Leu Ser Val lie Arg Lys lie Pro Ala Leu Thr Glu Asn AspPro GluThr Trp lie Val Cys Asn Phe Ser Val Asp His Asp Ser AlaP ro Leu Asn Asn Arg CysVal Arg Ala Lys lie Asn Val Ala Met IleCys Gin Thr Leu Val Ser Pro Pro Glu GlyAsn Gin Glu He Ser Arg(SEQ ID NO: 13) ro Leu Asn Asn Arg CysVal Arg Ala Lys lie Asn Val Ala Met IleCys Gin Thr Leu Val Ser Pro Pro Glu GlyAsn Gin Glu He Ser Arg (SEQ ID NO: 13)

[0229] Serl32A Cert突变体的核苷酸序列的核酸序列如SEQ ID NO: 14所示: [0229] the nucleotide sequence of a nucleic acid sequence mutants Serl32A Cert as SEQ ID NO: 14 below:

[0230] at gt cggata atcagagc tg gaactcgtcg ggc t cggagg aggat ccagagacggagtctgggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacattcatgggtggcaggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatctgaagatgaaacagagtatg getgcagagg atccatctgt cttagcaagg ctgtcatcacacctcacgattttgatgaa t gtcgatttga tattagtgta aatgatagtg tttggtatcttcgtgctcaggatccagatc atagacagca atggatagat gccattgaac agcacaagactgaatctggatatggatctg aatccagctt gcgtcgacat ggcgcaatgg tgtccctggtgtctggagcaagtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagtttacgtgagaagttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacacgctacagaagtactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaagggataaagtggtagaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttcttgcatagtaccaacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaatggtatagactttaaagggg aagcgataac ttttaaagca actactgetg gaatccttgcaacactttctcattgtattg aactaatggt taaacgtgag gacagctggc agaagagactggataaggaaactgagaaga aaagaagaac agaggaagca tataaaaatg caatgacagaacttaagaaaaaatcccact ttggaggacc agat [0230] at gt cggata atcagagc tg gaactcgtcg ggc t cggagg aggat ccagagacggagtctgggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacattcatgggtggcaggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatctgaagatgaaacagagtatg getgcagagg atccatctgt cttagcaagg ctgtcatcacacctcacgattttgatgaa t gtcgatttga tattagtgta aatgatagtg tttggtatcttcgtgctcaggatccagatc atagacagca atggatagat gccattgaac agcacaagactgaatctggatatggatctg aatccagctt gcgtcgacat ggcgcaatgg tgtccctggtgtctggagcaagtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagtttacgtgagaagttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacacgctacagaagtactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaagggataaagtggtagaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttcttgcatagtaccaacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaatggtatagactttaaagggg aagcgataac ttttaaagca actactgetg gaatccttgcaacactttctcattgtattg aactaatggt taaacgtgag gacagctggc agaagagactggataaggaaactgagaaga aaagaagaac agaggaagca tataaaaatg caatgacagaacttaagaaaaaatcccact ttggaggacc agat tatgaa gaaggcccta acagtctgattaatgaagaagagttctttg atgctgttga agctgctctt gacagacaag ataaaatagaagaacagtcacagagtgaaa aggtgagatt acattggcct acatccttgc cctctggagatgccttttcttctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaaccacatgacttactcattac aggatgtagg cggagatgcc aattggcagt tggttgtagaagaaggagaaatgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcctttaaaagctacccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctggaatgttgacgttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaacattagctgataatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctcagcgagacgtattatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccctgaaacttggatagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccgatgtgtccgtgccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccagagggaaaccaggaaattagca gggacaacat tctatgcaag attacatatg tagctaatgtgaaccctggaggatgggcac cagcctcagt gttaagggca gtggcaaagc gagagtatcctaaatttctaaaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag (SEQID N0:14)Serl32A Cert突变体的蛋白质序列如SEQ ID N0.15 tatgaa gaaggcccta acagtctgattaatgaagaagagttctttg atgctgttga agctgctctt gacagacaag ataaaatagaagaacagtcacagagtgaaa aggtgagatt acattggcct acatccttgc cctctggagatgccttttcttctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaaccacatgacttactcattac aggatgtagg cggagatgcc aattggcagt tggttgtagaagaaggagaaatgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcctttaaaagctacccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctggaatgttgacgttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaacattagctgataatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctcagcgagacgtattatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccctgaaacttggatagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccgatgtgtccgtgccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccagagggaaaccaggaaattagca gggacaacat tctatgcaag attacatatg tagctaatgtgaaccctggaggatgggcac cagcctcagt gttaagggca gtggcaaagc gagagtatcctaaatttctaaaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag (SEQID N0: 14) Serl32A Cert mutant protein sequence as SEQ ID N0.15 所示: Below:

[0231] Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp ProGlu ThrGlu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser LysTrp Thr Asn Tyr lie HisGly Trp Gin Asp Arg Trp Val Val Leu LysAsn Asn Ala Leu Ser Tyr Tyr Lys Ser GluAsp Glu Thr Glu Tyr GlyCys Arg Gly Ser lie Cys Leu Ser Lys Ala Val lie Thr ProHis AspPhe Asp Glu Cys Arg Phe Asp lie Ser Val Asn Asp Ser Val Trp TyrLeu ArgAla Gin Asp Pro Asp His Arg Gin Gin Trp lie Asp Ala IleGlu Gin His Lys Thr GluSer Gly Tyr Gly Ser Glu Ser Ser Leu ArgArg His Gly Ala Met Val Ser Leu Val SerGly Ala Ser Gly Tyr SerAla Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu ArgGlu LysLeu Ala Glu Met Glu Thr Phe Arg Asp lie Leu Cys Arg Gin Val AspThr LeuGin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys AspGlu Leu Gin Arg Asp LysVal Val Glu Asp Asp Glu Asp Asp Phe ProThr Thr Arg Ser Asp Gly Asp Phe Leu HisSer Thr Asn Gly Asn LysGlu Lys Leu Phe Pro His Val Thr Pro Lys Gly lie Asn Glylie AspPhe Lys Gly Glu Ala lie Thr Phe Lys Ala Thr Thr Ala Gly lie [0231] Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp ProGlu ThrGlu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser LysTrp Thr Asn Tyr lie HisGly Trp Gin Asp Arg Trp Val Val Leu LysAsn Asn Ala Leu Ser Tyr Tyr Lys Ser GluAsp Glu Thr Glu Tyr GlyCys Arg Gly Ser lie Cys Leu Ser Lys Ala Val lie Thr ProHis AspPhe Asp Glu Cys Arg Phe Asp lie Ser Val Asn Asp Ser Val Trp TyrLeu ArgAla Gin Asp Pro Asp His Arg Gin Gin Trp lie Asp Ala IleGlu Gin His Lys Thr GluSer Gly Tyr Gly Ser Glu Ser Ser Leu ArgArg His Gly Ala Met Val Ser Leu Val SerGly Ala Ser Gly Tyr SerAla Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu ArgGlu LysLeu Ala Glu Met Glu Thr Phe Arg Asp lie Leu Cys Arg Gin Val AspThr LeuGin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys AspGlu Leu Gin Arg Asp LysVal Val Glu Asp Asp Glu Asp Asp Phe ProThr Thr Arg Ser Asp Gly Asp Phe Leu HisSer Thr Asn Gly Asn LysGlu Lys Leu Phe Pro His Val Thr Pro Lys Gly lie Asn Glylie AspPhe Lys Gly Glu Ala lie Thr Phe Lys Ala Thr Thr Ala Gly lie LeuAla ThrLeu Ser His Cys lie Glu L eu Met Val Lys Arg Glu Asp SerTrp Gin Lys Arg Leu AspLys Glu Thr Glu Lys Lys Arg Arg Thr GluGlu Ala Tyr Lys Asn Ala Met Thr Glu LeuLys Lys Lys Ser His PheGly Gly Pro Asp Tyr Glu Glu Gly Pro Asn LeuAla ThrLeu Ser His Cys lie Glu L eu Met Val Lys Arg Glu Asp SerTrp Gin Lys Arg Leu AspLys Glu Thr Glu Lys Lys Arg Arg Thr GluGlu Ala Tyr Lys Asn Ala Met Thr Glu LeuLys Lys Lys Ser His PheGly Gly Pro Asp Tyr Glu Glu Gly Pro Asn

[0232] Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys IleGlu GluGin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr SerLeu Pro Ser Gly Asp AlaPhe Ser Ser Val Gly Thr His Arg Phe ValGin Lys Val Glu Glu Met Val Gin Asn HisMet Thr Tyr Ser Leu GinAsp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu GluGly GluMet Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly He Val Leu AspPro Leu LysAla Thr His Ala Val Lys Gly Val Thr Gly His Glu ValCys Asn Tyr Phe Trp Asn ValAsp Val Arg Asn Asp Trp Glu Thr ThrIle Glu Asn Phe His Val Val Glu Thr Leu AlaAsp Asn Ala lie IleIle Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin Arg AspValLeu Tyr Leu Ser Val lie Arg Lys lie Pro Ala Leu Thr Glu Asn AspPro Glu ThrTrp lie Val Cys Asn Phe Ser Val Asp His Asp Ser AlaPro Leu Asn Asn Arg Cys ValArg Ala Lys lie Asn Val Ala Met IleCys Gin Thr Leu Val Ser Pro Pro Glu Gly AsnGin Glu lie Ser ArgAsp Asn lie Leu Cys Lys lie Thr Tyr Val Ala Asn Val Asn ProGlyGly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu T [0232] Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys IleGlu GluGin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr SerLeu Pro Ser Gly Asp AlaPhe Ser Ser Val Gly Thr His Arg Phe ValGin Lys Val Glu Glu Met Val Gin Asn HisMet Thr Tyr Ser Leu GinAsp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu GluGly GluMet Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly He Val Leu AspPro Leu LysAla Thr His Ala Val Lys Gly Val Thr Gly His Glu ValCys Asn Tyr Phe Trp Asn ValAsp Val Arg Asn Asp Trp Glu Thr ThrIle Glu Asn Phe His Val Val Glu Thr Leu AlaAsp Asn Ala lie IleIle Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin Arg AspValLeu Tyr Leu Ser Val lie Arg Lys lie Pro Ala Leu Thr Glu Asn AspPro Glu ThrTrp lie Val Cys Asn Phe Ser Val Asp His Asp Ser AlaPro Leu Asn Asn Arg Cys ValArg Ala Lys lie Asn Val Ala Met IleCys Gin Thr Leu Val Ser Pro Pro Glu Gly AsnGin Glu lie Ser ArgAsp Asn lie Leu Cys Lys lie Thr Tyr Val Ala Asn Val Asn ProGlyGly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu T yrPro Lys PheLeu Lys Arg Phe Thr Ser Tyr Val Gin Glu Lys Thr AlaGly Lys Pro lie Leu Phe(SBQID NO:15) yrPro Lys PheLeu Lys Arg Phe Thr Ser Tyr Val Gin Glu Lys Thr AlaGly Lys Pro lie Leu Phe (SBQID NO: 15)

[0233] 人IgG抗体的ELISA检测 ELISA test [0233] human IgG antibody

[0234] 图2和图3分别显示了来自转染了人IgG modRNA的中国仓鼠卵巢(CHO)细胞和人胚肾(HEK,HER-2阴性)293细胞的酶联免疫实验(ELISA)。 [0234] Figures 2 and 3 show the (CHO) cells and human embryonic kidney derived from human IgG modRNA transfected Chinese Hamster Ovary (HEK, HER-2 negative) enzyme-linked immunoassays 293 cells (ELISA). 人胚肾(HEK) 293细胞在添加了购自Invitrogen的L-谷氨酰的⑶293培养基中培养到融合度达到80_90%。 Human embryonic kidney (HEK) 293 cells were grown in commercially available from Invitrogen of L- glutamyl ⑶293 medium to reach 80_90% confluency. CHO细胞在添加了L-谷氨酰、次黄嘌呤和胸腺嘧啶核苷的CD CHO培养基中培养。 CHO cells supplemented with L- glutamyl, CD CHO medium hypoxanthine and thymidine in the culture. 图2中,在盛有7ml培养基的购自Corning的75cm2培养皿中,用复合了购自Invitrogen的RNAiMax的24 μ gmodRNA转染2 X IO6个细胞。 In FIG. 2, in a petri dish filled with 7ml 75cm2 available from Corning, medium, with the compound from Invitrogen RNAiMax the transfection of 24 μ gmodRNA 2 X IO6 cells. 图3中,在24孔板中用复合了购自Invitrogen的RNAiMax的I μ g modRNA转染3,80,000个细胞。 In FIG 3, the composite of the 24 well plates were purchased from Invitrogen RNAiMax of I μ g modRNA 3,80,000 transfected cells. RNA = RNAiMAX复合体的制备:首先,将RNA在室温下与5 X体积稀释的⑶293培养基或⑶CHO培养基孵育10分钟;在第二个瓶中,将RNAiMAX试剂在室温下与IOX体积稀释的CD293培养基或CD CHO培养基孵育10分钟;随后,在添加到在滴状冷凝器中的细胞之前,将RNA瓶与RNAiMAX瓶混合并在室温下孵育20-30,然后以逐滴方式加入至细胞中。 RNA Preparation = RNAiMAX composite: First, the RNA was incubated with the diluted culture volume ⑶293 or 5 X ⑶CHO medium at room temperature for 10 min; In a second flask, diluted with IOX RNAiMAX reagent volume at room temperature CD293 CD CHO medium or medium for 10 min; then added to the cells prior to the condenser droplets, mixing with RNA RNAiMAX vial bottle and incubated at room temperature for 20-30, and then added in a dropwise manner to cells. 图2中,在转染后12、24、36小时测量培养基中的分泌人IgG的浓度。 In FIG. 2, the secretion of human 12,24,36 hours after transfection the medium was measured in the IgG concentration. 图3中,在36小时时测量分泌的人IgG。 In FIG. 3, measuring secreted human IgG in 36 hours. 培养物上清贮存在4摄氏度。 Culture supernatants were stored at 4 ° C. 按厂家说明使用购自Abcam的ELISA试剂盒测定自转染的人胚肾293细胞中分泌的曲妥珠单抗的量。 According to the manufacturer's instructions using commercially available from Abcam amount of an ELISA assay kit from transfected human embryonic kidney 293 cells secreted the trastuzumab. 这些数据显示了人源化IgG抗体(曲妥珠单抗)的modRNA (SEQ ID NOs: 6和7)能在人胚肾细胞中翻译,以及曲妥珠单抗被分泌到细胞外并释放到细胞外环境中。 These data show that modRNA humanized IgG antibody (trastuzumab) of (SEQ ID NOs: 6 and 7) can be translated in human embryonic kidney cells, and trastuzumab is secreted and released into the extracellular the extracellular environment. 进一步的,这些数据证明,以编码曲妥珠单抗的modRNA对细胞进行转染生产分泌蛋白可以规模放大至生物反应器或大型细胞培养条件。 Further, these data demonstrate that encoding of trastuzumab modRNA cells were transfected with the production of secreted proteins can be enlarged to the scale or large-scale bioreactor cell culture conditions.

[0235] modRNA产生的人I gG抗体的Western检测 [0235] produced human modRNA I gG Western detection antibody

[0236] 图4显示了以各I μ g的曲妥珠单抗重链和轻链的modRNA共转染CH0-K1细胞的Western Blot结果。 [0236] Figure 4 shows the Western Blot results of the respective I μ g modRNA trastuzumab heavy and light chains were co-transfected CH0-K1 cells. 为了检测蛋白质产物的翻译,按标准流程在24孔板中培养细胞,转染24小时后收集细胞上清或细胞裂解物并利用12%SDS-Page凝胶电泳进行分离,然后利用购自Invitrogen的iBlot转膜到硝化纤维膜上。 To detect translated protein product, cells were cultured according to the standard procedure in 24-well plates, cells were collected supernatants or cell lysates after transfection for 24 hours and using a 12% SDS-Page gel electrophoresis separation, and then using commercially available from Invitrogen iBlot transferred to a nitrocellulose membrane film. 在与缀合了DyLight® 594的抗人IgG抗体的兔多克隆抗体(ab96904, abcm, Cambridge, MA)以及缀合了碱性磷酸酶的抗Rb IgG的羊多克隆第二抗体孵育后,使用购自Invitrogen的Noves®碱性磷酸酶显色底物检测抗体。 In the DyLight® 594 conjugated anti-human IgG antibody after rabbit polyclonal antibody (ab96904, abcm, Cambridge, MA) and alkaline phosphatase conjugated goat polyclonal anti-Rb IgG secondary antibody incubation, using commercially available from Invitrogen Noves® alkaline phosphatase chromogenic substrate detection antibody.

[0237] modRNA产生的曲妥珠单抗和利妥昔单抗的细胞免疫染色 [0237] trastuzumab and rituximab immunostained cells produced modRNA

[0238] 图5显示了以曲妥珠单抗或利妥昔单抗的重链和轻链各500ng共转染CH0-K1细胞。 [0238] FIG. 5 shows in trastuzumab or rituximab heavy and light chains were cotransfected 500ng each CH0-K1 cells. 在购自Gibco的F-12K培养基和10%FBS中培养细胞。 Cultured cells were purchased from Gibco, and the culture medium of F-12K in 10% FBS. 用含4%聚甲醛的PBS溶液固定细胞并在室温下以0.l%Triton X-100的PBS溶液渗透5_10分钟。 Cells were fixed with PBS containing 4% paraformaldehyde in PBS and penetrate to 0.l% Triton X-100 at room temperature 5_10 minutes. 随后以室温PBS溶液洗涤细胞三次。 Then the cells were washed three times with PBS solution at room temperature. 用缀合了DyLight® 594的抗人IgG抗体的兔多克隆抗体(ab96904,abcm,Cambridge,MA)对曲妥珠单抗株和利妥昔单抗株进行染色。 Strains of trastuzumab and rituximab were stained with strain DyLight® 594 conjugated anti-human IgG antibody is rabbit polyclonal antibody (ab96904, abcm, Cambridge, MA). 用购自Invitrogen的DAPI染色剂对核DNA进行染色。 Nuclear DNA was stained with DAPI stain was purchased from Invitrogen. modRNA转染后,曲妥珠单抗和利妥昔单抗的蛋白质被翻译并定位在细胞I旲上。 ModRNA after transfection, trastuzumab and rituximab proteins are translated and positioned on the cell I Dae. 图片摄于转染后13小时。 Pictures taken at 13 hours post-transfection. [0239] modRNA产生的曲妥珠单抗和利妥昔单抗的结合免疫印迹实验 [0239] modRNA generated trastuzumab and rituximab binding western blot

[0240] 图6显示了曲妥珠单抗和利妥昔单抗的结合免疫印迹实验结果。 [0240] FIG. 6 shows the experimental results of trastuzumab and rituximab binding immunoblotting. 不同浓度的ErB2多妝(ab40048, abeam, Cambridge, MA) Different concentrations of ErB2 plurality makeup (ab40048, abeam, Cambridge, MA)

[0241] 针对曲妥珠单抗的抗原和CD20多肽(ab97360,abeam, Cambridge, MA)、针对利妥昔单抗的抗原以不同浓度(lOOng/μ I)在12%SDS-Page的凝胶上进行电泳并利用购自Invitrogen的iBlot转移到膜上。 [0241] For trastuzumab and CD20 antigen polypeptide (ab97360, abeam, Cambridge, MA), for rituximab at different concentrations of antigen (lOOng / μ I) gel in 12% SDS-Page electrophoresed using the iBlot available from Invitrogen, transferred to a membrane. 所述膜与其各自的细胞上清共孵育I小时,所述上清获自以曲妥珠单抗或利妥昔单抗的各500ng的重链和轻链共转染的CHO-Kl细胞。 The film with their respective cell supernatants were incubated I hour, to the supernatant obtained from trastuzumab or 500ng of each of the heavy and light chains rituximab co-transfected CHO-Kl cells. 所述膜用1%BSA封闭并添加缀合了碱性磷酸酶的抗人IgG第二抗体(abeam, Cambridge,MA)。 The membrane blocked with 1% BSA and added-conjugated anti-human IgG alkaline phosphatase second antibody (abeam, Cambridge, MA). 利用购自Invitrogen的Noves®碱性磷酸酶显色底物进行抗体检测。 Using commercially available from Invitrogen Noves® chromogenic substrate for alkaline phosphatase antibody. 该数据显示产生自modRNA的人源化的IgG抗体可以识别并结合其各自的抗原。 The data shows that people have from modRNA humanized IgG antibody can recognize and bind to their respective antigen.

[0242] 细胞增殖实验 [0242] Cell proliferation assay

[0243] SK-BR-3细胞系,来源于人乳腺癌的粘附细胞系,其过表达HER2/neu受体,可用于比较modRNA产生的曲妥珠单抗的抗增殖性质。 [0243] SK-BR-3 cell line, derived from human breast cancer cell line adhesion, overexpression of HER2 / neu receptor, can be used to compare the antiproliferative properties produced modRNA of trastuzumab. 可以向细胞培养物中添加不同浓度的产生自modRNA的纯化曲妥珠单抗和曲妥珠单抗,并通过一式三份的细胞毒性和存活力实验测定其对细胞生长的影响。 May be added to produce different concentrations of purified from the song modRNA trastuzumab, and trastuzumab to cell culture, and the experiment on cell growth determined by a formula cytotoxicity and viability triplicate.

[0244] SK0V-3肿瘤模型 [0244] SK0V-3 tumor model

[0245] modRNA产生的曲妥珠单抗的抗癌效果可以通过在28天的周期内向SK0V-3转基因小鼠中连续注射DmodRNA曲妥珠单抗,2)曲妥珠单抗,和3)modRNA曲妥珠单抗+modRNAGCSF测定。 [0245] modRNA produce anticancer effects of trastuzumab may be by continuous injection within the 28 day period SK0V-3 transgenic mice DmodRNA trastuzumab, 2) trastuzumab, and 3) modRNA trastuzumab + modRNAGCSF measured. 可以随时间监测肿瘤生长尺寸的减少。 Reduction in tumor size growth can be monitored over time.

[0246] 实施例4.过表达神经酰胺转移蛋白以提高治疗性抗体蛋白质在已建立的CHO细胞系中的生产能力。 [0246] Example 4. overexpressing ceramide transfer protein therapeutic antibodies to increase the protein production capability has been established CHO cell lines.

[0247] a)分批培养 [0247] a) batch cultures

[0248] 以单独的阳离子脂质递送介质(对照)或编码野生型神经酰胺转移蛋白(CERT)或无憐酸化能力(non-phosphorylation competent)的Serl32A CERT 突变体的合成mRNA转录本单次转染分泌人源化治疗性IgG抗体的产抗体CHO细胞系(CH0 DG44)。 [0248] Synthesis of mRNA transcripts in a single cationic lipid delivery vehicle (control) or encoding wild type ceramide transfer protein (the CERT) without pity or acidifying power (non-phosphorylation competent) Serl32A CERT mutant of this single transfection secreting humanised therapeutic antibody IgG antibody-producing CHO cell line (CH0 DG44). CERT是哺乳动物中的重要胞质蛋白,其将鞘磷脂神经酰胺自内质网转移到高尔基复合体,在此神经酰胺被转化成鞘脂(Hanada等,2003年)。 CERT is an important cytosolic protein in a mammal, which ceramide from sphingomyelin endoplasmic reticulum to the Golgi complex, where the ceramide is converted to sphingolipids (Hanada et al., 2003). CERT的过表达大大增强分泌蛋白向胞质膜的转移并改善通过分泌途径自真核细胞转运的蛋白质的生产,从而增强了蛋白质向培养基中的分泌。 CERT overexpression of a secreted protein greatly enhance the transfer of the plasma membrane and to improve the production of proteins through the secretory pathway from eukaryotic cell transport, thereby enhancing the secretion of the protein into the culture medium. 合成的mRNA转录本以2-5:1的载体:RNA比与阳离子脂质递送介质预混。 Synthesis of mRNA transcripts present in a 2-5: 1 vector: premix ratio of an RNA delivery media with cationic lipids. 起始接种浓度为约2X105活细胞/mL。 Initial inoculation concentration of about 2X105 viable cells / mL. 在指数培养生长期过程中在起始培养接种后,递送合成mRNA转录本以达到合成的mRNA的最终拷贝数量在每细胞10X IO2到10X IO3之间。 Culture in the exponential growth phase after initiation of culture during the inoculation, the delivery synthesized mRNA transcripts present in an amount to achieve a final synthesis of mRNA copies between each cell 10X IO2 to 10X IO3. 用于产生细胞接种物的所有阶段和生物反应器中的培养物生长的基础细胞培养基是包含谷氨酰、碳酸氢钠、胰岛素和甲氨蝶呤的改良⑶-CHO培养基。 Basal cell culture medium used to produce the cells of all stages of inoculum and bioreactor culture was grown glutamyl, sodium bicarbonate, insulin and methotrexate ⑶-CHO medium containing modified. 在添加全部组分后用IN HCl或IN NaOH将培养基的pH值调节到7.0。 The pH of the medium was adjusted to 7.0 using IN HCl or IN NaOH after addition of all the components. 在第7、14、21或28+天结束培养运行时间。 In the first training run time of 14, 21 or 28+ days end. 可以使用生产水平50L级的反应器(带有2个船用叶轮的不锈钢反应器)且可升级到> 10,000L不锈钢反应器(申请日为2002年12月23日的普通转让专利申请USSer.N0.60/436,050,和USSer.N0.10/740,645中所述)。 Production levels can 50L stage reactor (stainless steel reactor with two marine impellers) and may be upgraded to> Common Patent transfer 10,000L stainless steel reactor (filed December 23, 2002 Application USSer.N0 .60 / 436,050, and USSer.N0.10 / 740,645 described). 数据获取系统(Intellution Fix32)全程记录温度、pH值和溶解氧(D0)。 Data acquisition system (Intellution Fix32) throughout the recording temperature, pH and dissolved oxygen (D0). 气流通过旋转式流量计控制。 Air flow is controlled by rotameter. 空气通过液下玻璃料(孔径5 μ m)向反应器鼓气并通过反应器顶部空间移除C02。 Air was bubbled into the reactor and the gas is removed through the top of the C02 solution space of the reactor through the lower glass frit (pore size 5 μ m). 通过同样的玻璃料鼓入分子氧以进行DO控制。 By bubbling the same frit for DO control the molecular oxygen. CO2通过同样的玻璃料鼓泡鼓入以进行pH控制。 CO2 was bubbled through the same frit bubbling for pH control. 每天从反应器中移出细胞样品。 Day cell sample is removed from the reactor. 用于细胞计数的样品用台盼蓝(Sigma, St.Louis, M0.)染色。 Sample for cell count (. Sigma, St.Louis, M0) were stained with trypan blue. 使用显微通过细胞计数器进行细胞计数和细胞活力的测定。 It was measured using microscopic cell counting and viability by cell counter. 为了分析代谢物,以2000rpm(4°C )离心额外的样品20分钟以分离细胞。 For analysis of metabolites to 2000rpm (4 ° C) additional sample was centrifuged for 20 minutes to separate the cells. 分析上清的下述参数:效价、唾液酸、葡萄糖、乳酸盐、谷酰胺、谷氨酸盐、pH、p02, pC02,氨水和任选的乳酸脱氢酶(LDH)。 The supernatant is analyzed following parameters: titer, sialic acid, glucose, lactate, glutamine, glutamate, pH, p02, pC02, ammonia and, optionally, lactate dehydrogenase (LDH). 将额外的备份样品冷冻在_20°C。 The additional backup samples were frozen at _20 ° C. 为测定分泌的人源化IgG抗体的效价,自所有稳定细胞库的留种培养物获得上清,通过ELISA测量IgG效价并除以平均细胞数以计算特定生产能力。 To determine the secretion of the humanized IgG antibody titers from all stable cell pools Seed culture supernatant obtained, IgG titers were measured by ELISA and divided by the average number of cells to calculate the specific productivity. 最高值是有Serl32A CERT突变(SEQ ID N0.14)的细胞库,其次是野生型CERT (SEQ ID N0.12)。 The maximum value is Serl32A CERT mutant (SEQ ID N0.14) of the cell bank, followed by wild type CERT (SEQ ID N0.12). 相对于只有载体或未转染细胞,以上两者的IgG表达显著增强。 With respect to the vector or transfected cells only, IgG expression over both significantly enhanced.

[0249] b)连续或分批补料培养 [0249] b) continuous or fed-batch culture

[0250] 用单独的阳离子脂质递送介质(对照)或编码野生型神经酰胺转移蛋白或无磷酸化能力的Serl32A CERT突变的合成mRNA转录本转染分泌人源化IgG抗体的产抗体CHO细胞系(CHO DG44)。 [0250] a delivery vehicle (control) or a separate encoding wild-type cationic lipid ceramide transfer protein or no ability to phosphorylate a synthetic Serl32A CERT mutant mRNA transcript transfected humanized IgG antibody secreting antibody-producing CHO cell line (CHO DG44). 合成的mRNA转录本以2-5:1的载体:RNA比与阳离子脂质递送介质预混。 Synthesis of mRNA transcripts present in a 2-5: 1 vector: premix ratio of an RNA delivery media with cationic lipids. 起始接种浓度为约2X IO5活细胞/mL。 Initial inoculation concentration of about 2X IO5 viable cells / mL. 在指数培养生长期过程中在起始培养接种后,递送合成的mRNA转录本以达到合成mRNA的最终拷贝数量在每细胞IOX IO2到IOX IO3之间。 The final number of copies during the exponential growth phase culture after initial culture inoculation delivered synthetic mRNA transcript in order to achieve synthesis of mRNA per cell IOX IO2 between IOX IO3. 在产生细胞接种物的整个阶段以及在生物反应器中培养物的生长阶段所使用的基础细胞培养基是包含谷氨酰、重碳酸钠、胰岛素和甲氨蝶呤的改良CD-CHO培养基。 Basal cell culture medium at all stages of cell inoculum generation and the growth stage of the culture in the bioreactor was used containing glutamyl, sodium bicarbonate, insulin and methotrexate modified CD-CHO medium. 在添加全部组分后将培养基的PH值用IN HCl或IN NaOH调节到7.0。 All media components added after the PH value was adjusted to 7.0 using IN HCl or IN NaOH. 使用5L级生物反应器(带有一个船用叶轮的玻璃反应器)以获得最大的CERT蛋白产量和分泌人源化IgG抗体曲线。 5L bioreactor using stage (glass reactor with one marine impeller) to obtain the maximum yield of protein CERT and humanized IgG antibody secretion profile. 对于连续或分批补料培养,在运行期间,通过每天一次或多次(或持续地)以新鲜培养基补充培养物培养基而延长培养运行时间。 For continuous or fed-batch culture, during operation, by one or more times daily (or continuously) with fresh culture medium supplemented extended culture run time. 在连续或分批补料培养的方案中,培养物以持续地补充注入或其他添加到培养基的方式按时、定受控的和/或编程程序化的方式自动接受培养基,从而在培养物中达到和保持培养物中合成mRNA:载体的合适量。 In a continuous or fed-batch culture solution, the culture was supplemented continuously injected or otherwise added to the medium in time, given a controlled and / or programmed for automatic, programmed to accept the medium so that the culture was reached and maintained the culture mRNA synthesis: An appropriate amount of the carrier. 典型的方法是从开始进行培养到收获细胞的当天,在进行培养的每天推注给料一次包含合成的mRNA:载体的给料培养基的给料方式。 The typical approach is to culture the cells were harvested from the start of the day, every day during the culture bolus feed comprising a synthetic mRNA: a carrier feed media feed mode. 每日给料量记录在批次表格上。 Daily feeding amount recorded in the batch form. 使用生产水平50L级的反应器(带有2个船用叶轮的不锈钢反应器)且可升级到> 10,000L不锈钢反应器。 Use production level 50L stage reactor (stainless steel reactor with two marine impellers) and may be upgraded to> 10,000L stainless steel reactor. 数据获取系统(Intellution Fix32)全程记录`温度、pH值和溶解氧(DO)。 Data acquisition system (Intellution Fix32) `whole record of temperature, pH and dissolved oxygen (DO). 气流通过旋转式流量计控制。 Air flow is controlled by rotameter. 空气通过液下玻璃料(孔径5 μ m)鼓入反应器并通过反应器顶部空间移除C02。 Air through submerged frit (pore size 5 μ m) bubbled into the reaction and removing the headspace of the reactor through C02. 分子氧通过同样的玻璃料鼓入以进行DO控制。 Molecular oxygen by bubbling same frit for DO control. CO2通过同样的玻璃料鼓入以进行pH控制。 CO2 through the same frit bubbling for pH control. 每天从反应器中移出细胞样品。 Day cell sample is removed from the reactor. 用于细胞计数的样品一般用台盼蓝(Sigma, St.Louis, M0.)染色。 Samples used for general cell count with trypan blue (Sigma, St.Louis, M0.) Staining. 利用显微镜通过细胞计数器进行细胞计数和细胞活力的测定。 Cell counts and viability were determined by cell counter cells with a microscope. 为了分析代谢物,以2000rpm(4°C )离心额外的样品20分钟以分离细胞。 For analysis of metabolites to 2000rpm (4 ° C) additional sample was centrifuged for 20 minutes to separate the cells. 分析上清的下述参数:效价、唾液酸、葡萄糖、乳酸盐、谷酰胺、谷氨酸盐、pH、p02、pC02、氨水和任选的乳酸脱氢酶(LDH)。 The supernatant is analyzed following parameters: titer, sialic acid, glucose, lactate, glutamine, glutamate, pH, p02, pC02, ammonia and, optionally, lactate dehydrogenase (LDH). 额外的备份样品冷冻在_20°C。 Additional backup samples were frozen at _20 ° C. 为测定分泌的人源化IgG抗体的效价,自所有稳定细胞库的留种培养物获得上清,通过ELISA测量IgG效价并除以平均细胞数以计算特定生产能力。 To determine the secretion of the humanized IgG antibody titers from all stable cell pools Seed culture supernatant obtained, IgG titers were measured by ELISA and divided by the average number of cells to calculate the specific productivity. 最高值是有Serl32A CERT突变(SEQ ID N0.14)的细胞库,其次是野生型CERT (SEQ ID NO: 10或12)。 The maximum value is Serl32A CERT mutant (SEQ ID N0.14) of the cell bank, followed by wild type CERT (SEQ ID NO: 10 or 12). 相对于只有载体或未转染细胞,以上两者的IgG表达显著增强。 With respect to the vector or transfected cells only, IgG expression over both significantly enhanced.

[0251] 等价物和范围 [0251] equivalents and scope

[0252] 本领域技术人员可以仅使用常规实验认识到或能够确定本文所述的特定具体实施方式的多种等价物。 [0252] Those skilled in the art may be used alone more than routine experimentation, equivalents to recognize or be able to determine the specific embodiment described herein a particular embodiment. 本发明的范围并非意图限定到上述说明书,而是如随附的权利要求所示。 The scope of the present invention is not intended to be limited to the above description, but as shown in the appended claims.

[0253] 本领域技术人员可以仅使用常规实验认识到或能够确定按照本发明所公开的的具体实施方式的多种等价物。 [0253] Those skilled in the art using only routine experimentation can recognize, or be able to determine more equivalents to the specific embodiments disclosed in accordance with the present invention. 本发明的范围并非意图限定到上述说明书,而是如随附的权利要求所示。 The scope of the present invention is not intended to be limited to the above description, but as shown in the appended claims.

[0254] 权利要求书中,除非根据上下文有相反或其他的证据,所述“a”、“an”和“the”可以表示一个或多于一个。 [0254] appended claims, unless the context or other evidence to the contrary, the "a", "an" and "the" may mean one or more than one. 除非根据上下文有相反或其他证据,认为在一个或多个组的成员之间包含“或”的权利要求或说明是一个、多于一个或全部组成员存在于、参与或以其他方式关联于给定的产品或方法中。 Unless there is contrary or other evidence that between one or more members of the group comprising, "or" in the claims or is a description, more than one or all of the group members are present in, or otherwise associated with participation in the context to given product or process. 本发明包括只有组中的一员存在于、参与或以其他方式关联于给定的产品或方法中的具体实施方式。 The present invention includes only one group is present in, or otherwise associated with participating in a particular embodiment given product or process. 本发明包括超过一个或全部组的成员都存在于、参与或以其他方式关联于给定的产品或方法中的具体实施方式。 The present invention includes more than one or all of the group members are present in, or otherwise associated with participating in a particular embodiment given product or process. 进一步的,可以理解的是,本发明包括将一个或多个所列权利要求的一个或多个限制、元素、从句、描述术语等从引入另一个权利要求的所有的变形、组合和替换。 Further, it is understood that the present invention comprises one or more of the requirements set forth one or more limitations, elements, clauses, and other terms describing all modifications introduced from the other claims, combinations and substitutions. 例如,可以限定任何引用另一个权利要求的权利要求,使其包括一个或多个任意其他弓I用同一权利要求的权利要求中的限定。 For example, reference may be defined in any other claim claim, comprising one or more such that it defines any other bow of claim I with the same claims. 进一步的,除非另有说明或除非对于本领域普通技术人员而言出现反例或矛盾是显而易见的情况,则当权利要求叙及组合物,应当理解为包括出于本发明公开的任何目的的该组合物的使用方法,以及按照本发明公开的任意制备方法或本领域已知的其他方法以制备该组合物的方法。 Further, unless otherwise stated or unless there is a conflict or anti embodiments to those of ordinary skill in the situation apparent, when the classification and composition as claimed in claim should be understood to include any object of the present invention is disclosed for the compositions method was used, and any preparation method disclosed according to the present invention, or other methods known in the art methods for preparing the composition.

[0255] 当元素以列·表形式存在,例如,马库什形式,应当理解的是,同样公开了元素的每一个亚群,并且任何元素都可以自组中删除。 [0255] When elements are column-table form, e.g., in the form of Markush, it should be understood that the same is disclosed for each subgroup of elements, and any element may be deleted from the group. 应当理解的是,通常,当本发明或本发明的各方面被称为包含特定要素、特征等时,本发明或本发明各方面的某些具体实施方式由所述特定要素、特征等组成或基本由其组成。 It will be appreciated that, in general, when the present invention or aspects of the invention is referred to as comprising particular elements, features, etc., of the present invention or aspects of certain specific embodiments of the particular elements, features, etc., or consisting essentially of. 出于简明性目的,这些具体实施方式在本文中并没有从字词上做详细描述。 For purposes of simplicity, these embodiments and not described in detail herein from the word. 还应当注意到,术语“包含”是开放式的并且允许包含额外的组成成分或步骤。 It should also be noted that the term "comprising" is open-ended and allow comprise additional components or steps.

[0256] 给定的范围包括端值。 [0256] given range inclusive. 进一步地,应当理解,除非另有说明或根据上下文和本领域常规技术人员的理解另有证据,在本发明不同具体技术方案中,以范围表示的值可以假设成任意特定值或所述范围的子范围,除非上下文有清楚地其他指示,否则可以到范围下限的十分之一单位。 Further, it should be understood that, unless otherwise stated or the context and understanding of this art in accordance with the conventional art evidence of another, different particular aspect of the present invention, values ​​expressed as ranges can assume any specific value or to a range of sub-range, unless the context clearly otherwise directs, be possible to limit the scope of the tenth of the unit.

[0257] 此外,应当理解,任何落入现有技术的本发明的特定技术方案可以明确排除在任意一个或多个权利要求外。 [0257] Further, it should be understood that the particular aspect of the present invention falling within any of the prior art may be explicitly excluded from any one or more of the preceding claims. 因为这样的具体实施方式被认为是本领域普通技术人员已知的,即使该排除并未在本文中明确表明,其也可以被排除。 Because such specific embodiments are considered to be those of ordinary skill in the art, even if the exclusion was not clear that in this context, it may also be excluded. 不论是否与现有技术有关,可以基于任何原因从任意一个或多个权利要求中排除本发明组合物的任何特定具体实施方式(例如,任意的蛋白质、任意的核酸、任意的生产方法、任意的使用方法等)。 Whether or not related to the prior art, can be any reason to exclude any particular embodiment of the present invention is a composition from any one or more of the preceding claims (e.g., any protein, any nucleic acid, any of production methods, any methods of use).

[0258] 即使在引用中未明确表示,所有引用的来源,例如,参考文献、出版物、数据库、数据条目和本文引用的技术通过引用而引入到本申请。 [0258] Even if not explicitly illustrated in the references, the references to all sources, e.g., references, publications, databases, data entry techniques cited herein and incorporated by reference into the present application. 在引用的来源和本申请出现矛盾的情况下,应根据本申请的内容。 In the case cited sources in this application there is a conflict and should be in accordance with the present application.

[0259] 其他具体实施方式在权利要求中。 [0259] In another specific embodiment the claims.

Claims (31)

1.生产免疫球蛋白的试剂盒,所述试剂盒包含第一分离的核酸及其包装和说明书,所述第一分离的核酸含有i )编码免疫球蛋白的可翻译区和ii )核酸修饰,其中所述第一核酸能逃脱所述该第一分离的核酸所引入的细胞的天然免疫反应,其中所述可翻译区基本不含胞苷和尿嘧啶核苷。 1. Production of an immunoglobulin kit, said kit comprising a first isolated nucleic acid and packaging and instructions, said first isolated nucleic acid comprising i) encoding the immunoglobulin may be translated regions and ii) a modified nucleic acid, wherein the first nucleic acid can escape the natural immune response of the isolated nucleic acid introduced into the first cell, wherein said region is substantially free translation uridine and cytidine.
2.权利要求1所述的试剂盒,其中所述免疫球蛋白包含多肽,所述多肽选自由全长抗体、重链多肽、轻链多肽、Fab结构域或单链可变区片段(ScFv)多肽组成的组,所述第一分离的核酸包含信使核糖核酸,所述信使核糖核酸含有5-甲基-胞苷和假尿苷。 The kit according to claim 1, wherein said immunoglobulin comprises a polypeptide selected from the group consisting of the full length antibody, heavy chain polypeptide and light polypeptide chains, Fab domain, or single chain variable fragment (ScFv) the group of polypeptides, the nucleic acid comprises a first isolated mRNA, a mRNA containing 5-methyl - cytidine and pseudouridine.
3.权利要求1所述的试剂盒,其中所述第一分离的核酸包括选自下列组成组的核苷酸序列: a) SEQ ID N0:4和/或SEQ ID NO:6的核苷酸序列[利妥昔单抗]; b)与a)的核苷酸序列具有至少95%同一'丨生的核苷酸序列; c)编码SEQ ID N0:5和/或SEQ ID NO:7[利妥昔单抗]的多肽的核苷酸序列; d)与c)的核苷酸序列具有至少95%同一'丨生的核苷酸序列; e)编码与SEQ ID NO:5和/或SEQ ID NO:7[利妥昔单抗]具有至少95%同一性的多肽的核苷酸序列; f)与e)的核苷酸序列具有至少95%同一'丨生的核苷酸序列; g)包含a)_f)任一者的片段的核苷酸序列;以及h)a)-g)任一者的核苷酸序列的密码子优化变体。 The kit according to claim 1, wherein the first isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: nucleotides 6: a) SEQ ID N0: 4 and / or SEQ ID NO sequence [rituximab]; B) with a) a nucleotide sequence having a nucleotide sequence at least 95% identical to the 'raw Shu; c) encoding SEQ ID N0: 5 and / or SEQ ID NO: 7 [ rituximab] nucleotide sequence of the polypeptide; D) and c) a nucleotide sequence having a nucleotide sequence at least 95% identical to the 'raw Shu; E) encodes SEQ ID NO: 5 and / or SEQ ID NO: 7 [rituximab] polypeptide having a nucleotide sequence at least 95% identity; F) and e) a nucleotide sequence having a nucleotide sequence at least 95% raw Shu identical '; g) comprising a) _f) fragments of either nucleotide sequence; and h) a) -g) codon of the nucleotide sequence according to any one of the optimized variant.
4.权利要求3所述的试剂盒,其中所述免疫球蛋白免疫特异性结合到CD20多肽。 The kit according to claim 3, wherein the immunoglobulin polypeptide immunospecifically binds to CD20.
5.权利要求3所述的试剂盒,其中当与之接触时,所述免疫球蛋白特异性地诱导CD20+细胞的抗体依赖性细胞毒性。 The kit according to claim 3, wherein when in contact therewith, the immunoglobulin specifically induce antibody dependent cellular cytotoxicity CD20 + cells.
6.权利要求3所述的试剂盒,其中生产所述免疫球蛋白用于白血病、淋巴瘤和组织移植排异或自身免疫疾病或病症的治疗。 The kit according to claim 3, wherein the immunoglobulin produced in leukemia, lymphoma and tissue transplant rejection or autoimmune disease or condition being treated.
7.哺乳动物细胞,其通过使用权利要求1所述试剂盒产生。 7. A mammalian cell produced by using the kit of claim 1.
8.权利要求1所述的试剂盒,其中所述第一分离的核酸包括选自以下组成组的核苷酸序列: a) SEQ ID N0:8和/或SEQ ID NO: 10的核苷酸序列; b)与a)的核苷酸序列具有至少95%同一'丨生的核苷酸序列; c)编码SEQ ID NO:9和/或SEQ ID NO: 11的多肽的核苷酸序列; d)与c)的核苷酸序列具有至少95%同一'丨生的核苷酸序列; e)编码与SEQ ID NO:9和/或SEQ ID NO: 11具有至少95%同一性的多肽的核苷酸序列; f)与e)的核苷酸序列具有至少95%同一'丨生的核苷酸序列;以及g)包括a)_f)任一者的长度为至少30个核苷酸的片段的核苷酸序列。 8. The kit according to claim 1, wherein the first isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: a) SEQ ID N0: 8 and / or SEQ ID NO: 10 nucleotides sequence; b) with a) a nucleotide sequence having a nucleotide sequence at least 95% identical to the 'raw Shu; c) encoding SEQ ID NO: 11 is the nucleotide sequence of the polypeptide;: 9 and / or SEQ ID NO d) the nucleotide sequence of c) a nucleotide sequence having at least 95% identical to the 'raw Shu; E) encodes SEQ ID NO: 9 and / or SEQ ID NO: 11 having at least 95% identity to the polypeptide nucleotide sequence; F) and e) a nucleotide sequence having a nucleotide sequence at least 95% identical 'Shu green; and g) comprises a) _f) according to any one of the length of at least 30 nucleotides nucleotide sequence fragments.
9.权利要求8所述的试剂盒,其中所述免疫球蛋白免疫特异性结合到HER-2/neu受体多肽。 9. The kit according to claim 8, wherein the immunoglobulin specifically binds to the immune HER-2 / neu receptor polypeptide.
10.权利要求8所述的试剂盒,其中当与之接触时,所述免疫球蛋白特异性地诱导HER2/neu+细胞的抗体依赖性细胞毒性、细胞凋亡、细胞周期停滞或它们的组合。 10. The kit according to claim 8, wherein when in contact therewith, the immunoglobulin specifically HER2 neu + inducing antibody-dependent cellular cytotoxicity, apoptosis, cell cycle arrest / or combinations thereof.
11.权利要求8所述的试剂盒,其中生产所述免疫球蛋白用于HER2/neu+乳腺癌的治疗。 11. The kit according to claim 8, wherein the production of the immunoglobulin for the treatment of HER2 / neu + breast cancer.
12.分离的免疫球蛋白,其由自包含第一分离的核酸的生产细胞产生,所述第一分离的核酸含有i )编码免疫球蛋白的可翻译区和ϋ )核酸修饰,其中所述第一核酸能逃脱所述细胞的天然免疫反应,其中所述可翻译区基本不含胞苷或尿嘧啶核苷或者胞苷和尿嘧啶核苷的组合。 12. The isolated immunoglobulin, comprising a nucleic acid consisting of a first isolated from the production cells, the first isolated nucleic acid comprising i) encodes an immunoglobulin and can be translated region ϋ) nucleic acid modification, wherein said first a nucleic acid can escape the innate immune response of said cells, wherein said region is substantially free of translation, or cytidine and uridine, cytidine or uridine in combination.
13.权利要求12所述的蛋白,其中所述生产细胞是分离自人受试者的细胞。 13. The protein of claim 12, wherein said producer cell is a cell isolated from a human subject.
14.药物制剂,其包含有效量的权利要求13所述的蛋白。 14. A pharmaceutical formulation, which protein according to claim 13 comprising an effective amount of a.
15.药物制剂,其包含有效量的第一核酸,其中所述第一核酸含有i )编码免疫球蛋白的可翻译区和ii )核酸修饰,其中所述第一核酸显示出由细胞核酸酶引起的降解的减少并且能够逃脱所述第一核酸所引入的细胞的天然免疫反应,其中所述可翻译区基本不含胞苷和尿嘧啶核苷。 15. A pharmaceutical formulation comprising an effective amount of a first nucleic acid, wherein said first nucleic acid comprising i) encoding the immunoglobulin may be translated regions and ii) a nucleic acid modification, wherein the first nucleic acid exhibits caused by cellular nucleases reduced degradation of the innate immune response and can escape the first nucleic acid introduced into a cell, wherein said region is substantially free translation uridine and cytidine.
16.权利要求15所述的药物制剂,其中所述第一核酸显示出由细胞核酸酶引起的降解的减少。 16. A pharmaceutical formulation according to claim 15, wherein the first nucleic acid exhibits reduced degradation caused by cellular nucleases.
17.在细胞内生产感兴趣的异源蛋白质的方法,包括步骤: i )提供能翻译蛋白质的靶细胞;以及ii )在感兴趣的异源蛋白质在细胞内生产的条件下,将包含第一分离核酸的组合物引入靶细胞,其中所述第一分离的核酸含有编码所述感兴趣的异源蛋白质的可翻译区和核苷修饰。 17. A method of producing a heterologous protein of interest in a cell, comprising the steps of: i) providing a target cell capable of protein translation; and ii) under conditions producing heterologous protein of interest in a cell, comprising a first the isolated nucleic acid composition is introduced into a target cell, wherein the first isolated nucleic acid encoding a heterologous protein of interest can be translated and the region nucleoside modification.
18.权利要求17所述·的方法,进一步包括自所述细胞充分纯化感兴趣的蛋白质的步骤。 * 18. The method of claim 17, further comprising the step of the protein of interest is purified from the cell sufficiently.
19.权利要求17所述的方法,其中所述感兴趣的蛋白质是分泌蛋白。 19. The method of claim 17, wherein said protein of interest is a secreted protein.
20.权利要求17所述的方法,其中所述感兴趣的蛋白质是免疫球蛋白。 20. The method according to claim 17, wherein said protein of interest is an immunoglobulin.
21.权利要求17所述的方法,其中所述感兴趣的蛋白质是细胞内蛋白。 21. The method of claim 17, wherein said protein of interest is an intracellular protein.
22.增加细胞内感兴趣的重组表达蛋白质的产量的方法,包括步骤: i )提供包含编码感兴趣的蛋白质的异源核酸的靶细胞;以及ii )在所述效应蛋白在细胞内生产的条件下,将包含第一分离核酸的组合物引入靶细胞,从而提高细胞内重组表达蛋白质的产量,其中所述第一分离的核酸含有编码翻译效应蛋白的可翻译区和核苷修饰。 22. A method of increasing interest within cells expressing the recombinant production of protein, comprising the steps of: i) providing a protein of interest comprising a heterologous nucleic acid encoding a target cell; and ii) conditions for the production in the cell of the effector proteins under composition, comprising a first isolated nucleic acid into a target cell, thereby increasing the intracellular expression of recombinant protein production, wherein the first isolated nucleic acid that encodes an effector protein may be translated and untranslated region of nucleoside modification.
23.权利要求22所述的方法,其中所述感兴趣的蛋白质是免疫球蛋白。 23. The method according to claim 22, wherein said protein of interest is an immunoglobulin. 权利要求22所述的方法,其中所述感兴趣的蛋白质是分泌蛋白。 The method according to claim 22, wherein said protein of interest is a secreted protein.
24.权利要求22所述的方法,其中所述感兴趣的蛋白质是细胞内蛋白。 24. The method according to claim 22, wherein said protein of interest is an intracellular protein.
25.权利要求22所述的方法,其中所述靶细胞是哺乳动物细胞。 25. The method according to claim 22, wherein said target cell is a mammalian cell.
26.权利要求22所述的方法,其中所述靶细胞是酵母细胞。 26. The method of claim 22, wherein said target cell is a yeast cell.
27.权利要求22所述的方法,其中所述靶细胞是细菌细胞、昆虫细胞或植物细胞。 27. The method according to claim 22, wherein the target cell is a bacterial cell, insect cell or plant cell.
28.权利要求22所述的方法,其中所述翻译效应蛋白是神经酰胺转移蛋白(CERT)。 28. The method according to claim 22, wherein said protein is a translation effector ceramide transfer protein (CERT).
29.调节靶细胞内感兴趣蛋白质的水平的方法,包括步骤: i )调节靶细胞内至少一种翻译效应分子的活性,其中所述调节包括将含有编码翻译效应蛋白的可翻译区和核苷修饰的第一分离的核酸引入靶细胞;以及ii )培养所述细胞。 29. A method of adjusting the level of a protein of interest in a target cell, comprising steps: i) modulating the activity of at least one translation effector molecules within a target cell, wherein said adjusting comprises encoding the translated region containing the effector proteins and the translated nucleotide modified first isolated nucleic acid into a target cell; and ii) culturing the cell.
30.用于蛋白质生产的试剂盒,包括编码可翻译区的第一分离的核酸及其包装和说明书,其中所述可翻译区编码蛋白质,其中所述第一核酸含有核酸修饰,其中与不含核酸修饰的核酸相比,所述第一核酸在所述第一分离核酸所引入的细胞内显示出降解的减少。 30. A kit for producing a protein, comprising a first isolated nucleic acid encoding a translation region and their packaging and instructions, wherein the translatable region encoding a protein, wherein said first nucleic acid contains a modified nucleic acid, which does not contain nucleic acid modifications as compared to a nucleic acid, said first nucleic acid exhibit reduced degradation within the first separation cell the introduced nucleic acid.
31.用于蛋白质生产的试剂盒,包括编码可翻译区的第一分离的核酸及其包装和说明书,其中所述可翻译区编码蛋白质,其中所述第一核酸含有核酸修饰,其中与不含核酸修饰的核酸相比,所述第一核酸显示出在所述第一分离的核酸所引入的细胞中更稳定。 31. A kit for producing a protein, comprising a first isolated nucleic acid encoding a translation region and their packaging and instructions, wherein the translatable region encoding a protein, wherein said first nucleic acid contains a modified nucleic acid, which does not contain nucleic acid modifications as compared to a nucleic acid, said first nucleic acid was more stable in the first cell of the isolated nucleic acid of the introduced.
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